CA2162557C - Conserved motif of hepatitis c virus e2/ns1 region - Google Patents
Conserved motif of hepatitis c virus e2/ns1 region Download PDFInfo
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- CA2162557C CA2162557C CA002162557A CA2162557A CA2162557C CA 2162557 C CA2162557 C CA 2162557C CA 002162557 A CA002162557 A CA 002162557A CA 2162557 A CA2162557 A CA 2162557A CA 2162557 C CA2162557 C CA 2162557C
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- hcv
- amino acid
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- antibodies
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Classifications
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
- C07K16/109—Hepatitis C virus; Hepatitis G virus
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- A—HUMAN NECESSITIES
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- A61K38/00—Medicinal preparations containing peptides
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/24011—Flaviviridae
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- C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
- Y10S530/806—Antigenic peptides or proteins
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
- Y10S530/82—Proteins from microorganisms
- Y10S530/826—Viruses
Abstract
The hypervariable region (E2HV) of the putative hepatitis C virus (HCV) glycoprotein F2/NS1, between about amino acid 384 to about amino acid 414, is a rapidly evolving region of HCV, and is likely to be under positive immune selection. A newly discovered motif within this hypervariable region is immunogenic and conserved with respect to the character of the amino acids. In many isolates, this motif falls between amino acids 401 to 406 or 407. The discovery of this motif allows for additional materials and methods to treat and diagnose HCV.
Description
216~~~"~
O 94126306 PCTIUS94l04853 Description Technical Field This invention relates generally to the field of hepatitis C virus (HCV) and, more specifically, to the discovery of an immunologically important motif in the E2/NS 1 region.
Background Hepatitis C virus (EiCV) has been identified as the major causative agent of post-transfusion non-A, non-B hepatitis (NANBH). Materials and methods for obtaining the viral genomic sequences are known. See, e.g., PCT Publ. Nos.
W089104669, W090/11089, and WO90/14436. For general information about HCV, see Houghton et al., Hepatology (1991) 14:381-388.
Molecular characterization of the HCV genome indicates that it is a RNA
molecule of positive polarity containing approximately 9,500 nucleotides comprising a long translational open-reading frame (ORF) that could encode a large polypeptide of approximately 3000 amino acids (aa) beginning with the first in-frame methionine codon. A hypervariable domain located at the amino terminus of the putative envelope glycoprotein E2/NS 1 (also called E2) has been located, see PCT Publ. N~o. W093/016126; Weiner et al., Virolow (1991) 180:842-848; Weiner et a:l., Proc. Natl. Acad. Sci. USA (1992) 89:3468-3472;
Weiner et al., Vaccines 9:2:303-308, Cold Spring Harbor Laboratory.
As observed for other RNA viruses, there is a substantial fluidity of the HCV genome resulting from an error-prone replicase and the absence of repair mechanisms that operate during DNA replication. Even in a single infected individual, the HCV genome does not exist as a homogeneous species. Rather, it exists as a quasi-species distribution of closely related but nevertheless heterogeneous genomes. Martell et al., J. Virol. (1992) 66:3225-3229. In addition, the process of host selection and adaptation of a rapidly mutating genome has led to the evolution of many distinct (yet still fluid) HCV genotypes. At least WO 94126306 PCTlUS94l04853 four different HCV genotypes can be distinguished according to the actual degree of nucleotide and amino acid relatedness of full length sequences, and additional different genotypes have been identified based on partial sequences. Mori et al. , Biochem. Biophys. Res. Comm. (1992) 183:334-342; Chan et al., J. Gen. Virol.
(1992) 73:1131; Cha et al., Proc. Natl. Acad. Sci. USA (1992) 89:7144-7148.
Disclosure of the Invention The present invention is directed to novel vaccine strategies for the treatment of HCV infection and assays for the diagnosis of HCV.
The hypervariable region of E2/NS 1 (E2HV) between about amino acid 384 to about amino acid 414 is a rapidly evolving region of HCV and appears to be under positive immune selection. The present invention relates to the existence within this subregion of a motif that is immunogenic and conserved with respect to the character of the amino acids. Although the E2HV amino acid sequences need not be identical within this motif, a definite pattern exists. In HCV 1, as well as a number of other isolates, this motif is seen at about amino acids 401 to 407.
The presence of this motif in an immunogenic polypeptide is detectable by antibody binding.
The discovery of this motif within the E2/NS1 hypervariable region allows for a strategy of producing materials, including polypeptides and antibodies that may be used for treatment of HCV, whether by direct or passive immunization.
Additionally, diagnostic methods employing immunoassays or nucleic acid assays are included herein.
Thus, in one aspect of this invention, a method for passively immunizing an individual for treatment of hepatitis C virus (HCV) infection is provided, the method comprising administering to the individual an antibody composition comprising an antibody capable of binding to a motif comprising an amino acid sequence aal-aa2-aa3-aa4-aa5-aa6 wherein aal is S, G, A, D, K, R or T; aa2 is L, F, I, M or W; aa3 is F or L;
aa4 is any amino acid; aa5 is any amino acid; and aa6 is G or A. In a further embodiment, aa7 is present and attached to aa6; aa7 is A, P, or S.
O 94126306 PCTIUS94l04853 Description Technical Field This invention relates generally to the field of hepatitis C virus (HCV) and, more specifically, to the discovery of an immunologically important motif in the E2/NS 1 region.
Background Hepatitis C virus (EiCV) has been identified as the major causative agent of post-transfusion non-A, non-B hepatitis (NANBH). Materials and methods for obtaining the viral genomic sequences are known. See, e.g., PCT Publ. Nos.
W089104669, W090/11089, and WO90/14436. For general information about HCV, see Houghton et al., Hepatology (1991) 14:381-388.
Molecular characterization of the HCV genome indicates that it is a RNA
molecule of positive polarity containing approximately 9,500 nucleotides comprising a long translational open-reading frame (ORF) that could encode a large polypeptide of approximately 3000 amino acids (aa) beginning with the first in-frame methionine codon. A hypervariable domain located at the amino terminus of the putative envelope glycoprotein E2/NS 1 (also called E2) has been located, see PCT Publ. N~o. W093/016126; Weiner et al., Virolow (1991) 180:842-848; Weiner et a:l., Proc. Natl. Acad. Sci. USA (1992) 89:3468-3472;
Weiner et al., Vaccines 9:2:303-308, Cold Spring Harbor Laboratory.
As observed for other RNA viruses, there is a substantial fluidity of the HCV genome resulting from an error-prone replicase and the absence of repair mechanisms that operate during DNA replication. Even in a single infected individual, the HCV genome does not exist as a homogeneous species. Rather, it exists as a quasi-species distribution of closely related but nevertheless heterogeneous genomes. Martell et al., J. Virol. (1992) 66:3225-3229. In addition, the process of host selection and adaptation of a rapidly mutating genome has led to the evolution of many distinct (yet still fluid) HCV genotypes. At least WO 94126306 PCTlUS94l04853 four different HCV genotypes can be distinguished according to the actual degree of nucleotide and amino acid relatedness of full length sequences, and additional different genotypes have been identified based on partial sequences. Mori et al. , Biochem. Biophys. Res. Comm. (1992) 183:334-342; Chan et al., J. Gen. Virol.
(1992) 73:1131; Cha et al., Proc. Natl. Acad. Sci. USA (1992) 89:7144-7148.
Disclosure of the Invention The present invention is directed to novel vaccine strategies for the treatment of HCV infection and assays for the diagnosis of HCV.
The hypervariable region of E2/NS 1 (E2HV) between about amino acid 384 to about amino acid 414 is a rapidly evolving region of HCV and appears to be under positive immune selection. The present invention relates to the existence within this subregion of a motif that is immunogenic and conserved with respect to the character of the amino acids. Although the E2HV amino acid sequences need not be identical within this motif, a definite pattern exists. In HCV 1, as well as a number of other isolates, this motif is seen at about amino acids 401 to 407.
The presence of this motif in an immunogenic polypeptide is detectable by antibody binding.
The discovery of this motif within the E2/NS1 hypervariable region allows for a strategy of producing materials, including polypeptides and antibodies that may be used for treatment of HCV, whether by direct or passive immunization.
Additionally, diagnostic methods employing immunoassays or nucleic acid assays are included herein.
Thus, in one aspect of this invention, a method for passively immunizing an individual for treatment of hepatitis C virus (HCV) infection is provided, the method comprising administering to the individual an antibody composition comprising an antibody capable of binding to a motif comprising an amino acid sequence aal-aa2-aa3-aa4-aa5-aa6 wherein aal is S, G, A, D, K, R or T; aa2 is L, F, I, M or W; aa3 is F or L;
aa4 is any amino acid; aa5 is any amino acid; and aa6 is G or A. In a further embodiment, aa7 is present and attached to aa6; aa7 is A, P, or S.
~ 1 X25 In another aspect of this invention, an antibody capable of recognizing an antigenic determinant is provided, wherein the antigenic determinant comprises the amino acid sequence aal-aa2-aa3-aa4-aa5-aa6 wherein aal is S, G, A, D, K, R or T; aa2 is L, F, I, M or W; aa3 is F or L;
aa4 is any amino acid; aa5 is any amino acid; and aa6 is G or A. In a further embodiment, aa7 is present and attached to a.a6; aa7 is A, P, or S.
In a further aspect of this invention, an immunogenic polypeptide is provided comprising a motif characterized by aal-aa2-aa3-aa4-aa5-aa6 wherein aa1 is S, G, A, D, K, R or T; aa2 is L, F, I, M or VAT; aa3 is F or L;
aa4 is any amino acid; aa5 is any amino acid; and aa6 is G or A, provided that the motif is not contained within a 31 amino acid sequence of a naturally-occurring E2HV domain of an HCV isolate known as of May 1.2, 1993. In a further embodiment, aa7 is present and attached to aa6; aa7 is A, P, or S.
In a still further ~ispect of this invention, a vaccine is provided comprising: (1) at least one immunogenic polypeptide comprising a motif characterized by aal-aa2-aa3-aa4-aa5-aa6 wherein aal is S, G, A, D, K, R or T; aa2 is L, F, I, M or W; aa3 is F or L;
aa4 is any amino acid; aa5 is any aJnino acid; and aa6 is G or A; and (2) a pharmaceutically acceptable carrier.
In yet another aspect of this invention, a method of treating an individual for HCV infection is provided, the method comprising administering to the individual the vaccine as described abcve.
In another aspect of this invention, an immunoassay method for detecting anti-hepatitis C virus (HCV) antibodies in biological samples provided, the method comprising: (a) incubati~lg an antibody-containing biological sample suspected of containing anti-HCV antibodies with a probe antigen comprising an immunogenic polypeptide as described. above to permit the formation of an antibody-antigen complex; and (b) detecting the antibody-antigen complex containing the probe antigen.
KM"Tr .3a According to one aspect of the invention, there is provided an immunogenic poiypeptide which is a ;31 mer having the consensus sequence .T.VTGG.AARTT.G..SLF..G.SQ.IQL.I derived from the E2HV hypervariable region of HCV
or a truncated version thereof which retains the SI_F..G motif or variants thereof which retain the SLF..G motif and wh~;rein at one or more amino acid residue positions the amino acid residue is substituted by another amino acid residue or by a non-naturally occurring analogue thereof, whereby immunoreactivity of said truncated versions or' variants is maintained.
to According to another aspect of the invention, there is provided an immunoassay method for detecting anti-hepatitis ~; virus (HCV) antibodies in a biological sample, the method comprising the steps of:
(a) incubating an antibody - containing biological sample suspected of containing anti-HCV antibodies with an immunogenic polypeptide as claimed in any one of claims 1, ~, 3 and 8 to permit the formation of an antibody-antigen complex and (b) detecting whether any antibody-antigen complex containing said immunogenic polypeptide is formed. - -,_. ..._._r_.~_.m._.
.' r.., . . M "". "~..w * ..-. ".. " -*,.w. *"...*.n,.~ ,.. ,*.~ .. .w- ~~* .,uw*
,**~*~~..* ~~",""*,*"~", . .. ~~~*,~ ~.",*.~ ,""".,.,.,* , ~, ~" .~ .". ~, w ~.a ~a.-w . M... . ,w. "~,Mw. ." . . ...
aa4 is any amino acid; aa5 is any amino acid; and aa6 is G or A. In a further embodiment, aa7 is present and attached to a.a6; aa7 is A, P, or S.
In a further aspect of this invention, an immunogenic polypeptide is provided comprising a motif characterized by aal-aa2-aa3-aa4-aa5-aa6 wherein aa1 is S, G, A, D, K, R or T; aa2 is L, F, I, M or VAT; aa3 is F or L;
aa4 is any amino acid; aa5 is any amino acid; and aa6 is G or A, provided that the motif is not contained within a 31 amino acid sequence of a naturally-occurring E2HV domain of an HCV isolate known as of May 1.2, 1993. In a further embodiment, aa7 is present and attached to aa6; aa7 is A, P, or S.
In a still further ~ispect of this invention, a vaccine is provided comprising: (1) at least one immunogenic polypeptide comprising a motif characterized by aal-aa2-aa3-aa4-aa5-aa6 wherein aal is S, G, A, D, K, R or T; aa2 is L, F, I, M or W; aa3 is F or L;
aa4 is any amino acid; aa5 is any aJnino acid; and aa6 is G or A; and (2) a pharmaceutically acceptable carrier.
In yet another aspect of this invention, a method of treating an individual for HCV infection is provided, the method comprising administering to the individual the vaccine as described abcve.
In another aspect of this invention, an immunoassay method for detecting anti-hepatitis C virus (HCV) antibodies in biological samples provided, the method comprising: (a) incubati~lg an antibody-containing biological sample suspected of containing anti-HCV antibodies with a probe antigen comprising an immunogenic polypeptide as described. above to permit the formation of an antibody-antigen complex; and (b) detecting the antibody-antigen complex containing the probe antigen.
KM"Tr .3a According to one aspect of the invention, there is provided an immunogenic poiypeptide which is a ;31 mer having the consensus sequence .T.VTGG.AARTT.G..SLF..G.SQ.IQL.I derived from the E2HV hypervariable region of HCV
or a truncated version thereof which retains the SI_F..G motif or variants thereof which retain the SLF..G motif and wh~;rein at one or more amino acid residue positions the amino acid residue is substituted by another amino acid residue or by a non-naturally occurring analogue thereof, whereby immunoreactivity of said truncated versions or' variants is maintained.
to According to another aspect of the invention, there is provided an immunoassay method for detecting anti-hepatitis ~; virus (HCV) antibodies in a biological sample, the method comprising the steps of:
(a) incubating an antibody - containing biological sample suspected of containing anti-HCV antibodies with an immunogenic polypeptide as claimed in any one of claims 1, ~, 3 and 8 to permit the formation of an antibody-antigen complex and (b) detecting whether any antibody-antigen complex containing said immunogenic polypeptide is formed. - -,_. ..._._r_.~_.m._.
.' r.., . . M "". "~..w * ..-. ".. " -*,.w. *"...*.n,.~ ,.. ,*.~ .. .w- ~~* .,uw*
,**~*~~..* ~~",""*,*"~", . .. ~~~*,~ ~.",*.~ ,""".,.,.,* , ~, ~" .~ .". ~, w ~.a ~a.-w . M... . ,w. "~,Mw. ." . . ...
Brief Descn_ption of the Drawings Fig. 1 is a schematic of the genetic organization of HCV.
Fig. 2 shows the E2HV sequences for 90 HCV isolates.
Fig. 3 shows the H(;V E2HV sequence data from patients followed sequentially after HCV infection.
Fig. 4 shows the percent of conservation for each amino acid at positions 384 to 407 of E2HV.
Fig. 5 presents bar l;raphs of epitope mapping showing the binding of serum from sheep irnmuniz~ed with a peptide that spanned HCV 1 E2HV region to 8-mer overlapping mimotopes that spanned the same region.
Fig. 6 presents bar l;raphs of epitope mapping showing the binding of monoclonal anti-thyroxin antibodies to overlapping peptides of the E2HV
region.
Fig. 7 presents bar l;raphs of epitope mapping showing the binding of human serum albumin, pre~~lbumin, and TBG to overlapping peptides of the E2HV
region.
Modes for Can '"~m~ Out the Invention A. Definitions The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and imrnunology, which are within the skill of the art. Such techniques are explained fu',lly in the literature. See e.g., Sambrook, et al., MOLECULAR CLONING:, A LABORATORY MANUAL, SECOND EDITION
(1989); DNA CLONING, VOLUMES I AND II (D.N. Glover ed. 1985);
OLIGONUCLEOTIDE SYNTHESIS (M.J. Gait ed, 1984); NUCLEIC ACID
HYBRIDIZATION (B.D. Hames & S.J. Higgins eds. 1984); TRANSCRIPTION
AND TRANSLATION (B.ID. Hames & S.J. Higgins eds. 1984); ANIMAL CELL
CULTURE (R.I. Freshney ed. 1986); IIVIIvIOBILIZED CELLS AND ENZYMES
(IRL Press, 1986); B. Perb.al, A PRACTICAL GUIDE TO MOLECULAR
CLONING (1984); the series, METHODS IN ENZYMOLOGY (Academic Press, Inc.); GENE TRANSFER 'SECTORS FOR MAMMALIAN CELLS (J.H. Miller and M.P. Calos eds. 1987, Cold Spring Harbor Laboratory); Methods in 216;?55 Enzymology Vol. 154 and Vol. 155 (Wu and Gmssman, and Wu, eds., respectively); Mayer and Walker, eds. (1987), IMMLTNOCHF.IvECAL METHODS
IN CELL AND MOLECLfLAR BIOLOGY (Academic Press, London); Scopes, (1987), PROTEIN PLTRIF(CATTON: PRINCIPLES AND PRACTICE, Second 5 Edition (Springer-Verlag, :hT.Y.); and HANDBOOK OF EXPE;F;IIV>EN'TAL IM-MUNOLOGY, VOLiJME;S I-IV (D:M. Weir and C. C. BiaclcweU eds 1986).
Standard abbreviations for nucleotides and amino acids are used in this specification.
Hepatitis C virus (HCV) is a new member of the Family Flaviviridae, which includes the pestiviruses (hog cholera virus and bovine diarrhea virus) and the flaviviruses, examples ~of which are dengue and yellow fever virus. A
scheme of the genetic organization of HCV is shown in Fig. 1. Similar to the ftavi-and pestiviruses, HCV appears to encode a basic polypeptide domain ("C") at the N-terminus of the viral polyprotein follawed by two glycoprotein domains ("El,"
"E2/NS 1 ") upstream of the: nonstructural genes NS2 through NSS. The amino acid coordinates of the puLidve pratein domains are shown in Table 1.
Table 1. The Putative Protein Domains in HCV
Amino Acid Coordinates (approximate) Protein 192 - 383 El Because the El and E2lNS I regions of the genome encode putative envelope type glycoproteins, these regions are of particular interest with respect to immunogenicity and treatment of HCV .
Fig. 2 shows the E2HV sequences for 90 HCV isolates.
Fig. 3 shows the H(;V E2HV sequence data from patients followed sequentially after HCV infection.
Fig. 4 shows the percent of conservation for each amino acid at positions 384 to 407 of E2HV.
Fig. 5 presents bar l;raphs of epitope mapping showing the binding of serum from sheep irnmuniz~ed with a peptide that spanned HCV 1 E2HV region to 8-mer overlapping mimotopes that spanned the same region.
Fig. 6 presents bar l;raphs of epitope mapping showing the binding of monoclonal anti-thyroxin antibodies to overlapping peptides of the E2HV
region.
Fig. 7 presents bar l;raphs of epitope mapping showing the binding of human serum albumin, pre~~lbumin, and TBG to overlapping peptides of the E2HV
region.
Modes for Can '"~m~ Out the Invention A. Definitions The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and imrnunology, which are within the skill of the art. Such techniques are explained fu',lly in the literature. See e.g., Sambrook, et al., MOLECULAR CLONING:, A LABORATORY MANUAL, SECOND EDITION
(1989); DNA CLONING, VOLUMES I AND II (D.N. Glover ed. 1985);
OLIGONUCLEOTIDE SYNTHESIS (M.J. Gait ed, 1984); NUCLEIC ACID
HYBRIDIZATION (B.D. Hames & S.J. Higgins eds. 1984); TRANSCRIPTION
AND TRANSLATION (B.ID. Hames & S.J. Higgins eds. 1984); ANIMAL CELL
CULTURE (R.I. Freshney ed. 1986); IIVIIvIOBILIZED CELLS AND ENZYMES
(IRL Press, 1986); B. Perb.al, A PRACTICAL GUIDE TO MOLECULAR
CLONING (1984); the series, METHODS IN ENZYMOLOGY (Academic Press, Inc.); GENE TRANSFER 'SECTORS FOR MAMMALIAN CELLS (J.H. Miller and M.P. Calos eds. 1987, Cold Spring Harbor Laboratory); Methods in 216;?55 Enzymology Vol. 154 and Vol. 155 (Wu and Gmssman, and Wu, eds., respectively); Mayer and Walker, eds. (1987), IMMLTNOCHF.IvECAL METHODS
IN CELL AND MOLECLfLAR BIOLOGY (Academic Press, London); Scopes, (1987), PROTEIN PLTRIF(CATTON: PRINCIPLES AND PRACTICE, Second 5 Edition (Springer-Verlag, :hT.Y.); and HANDBOOK OF EXPE;F;IIV>EN'TAL IM-MUNOLOGY, VOLiJME;S I-IV (D:M. Weir and C. C. BiaclcweU eds 1986).
Standard abbreviations for nucleotides and amino acids are used in this specification.
Hepatitis C virus (HCV) is a new member of the Family Flaviviridae, which includes the pestiviruses (hog cholera virus and bovine diarrhea virus) and the flaviviruses, examples ~of which are dengue and yellow fever virus. A
scheme of the genetic organization of HCV is shown in Fig. 1. Similar to the ftavi-and pestiviruses, HCV appears to encode a basic polypeptide domain ("C") at the N-terminus of the viral polyprotein follawed by two glycoprotein domains ("El,"
"E2/NS 1 ") upstream of the: nonstructural genes NS2 through NSS. The amino acid coordinates of the puLidve pratein domains are shown in Table 1.
Table 1. The Putative Protein Domains in HCV
Amino Acid Coordinates (approximate) Protein 192 - 383 El Because the El and E2lNS I regions of the genome encode putative envelope type glycoproteins, these regions are of particular interest with respect to immunogenicity and treatment of HCV .
The average rate of change of the HCV genome within a single persistently-infected individual has been estimated to be 1-2 x 10-3 nt changes per site per year. However, there is a much higher rate of change at the extreme 5'-terminus of the gene encoding the N-terminus of the E2lNS l glycoprotein.
Weiner et al., in FRONTIERS IN VIROLOGY: DIAGNOSIS OF HUMAN
VIRUSES BY POLYMER~rSE CHAIN REACTION TECHNOLOGY (Springer Verlag, Heidelberg, 1992). This E2 hypervariable region (E2HV) spanning amino acids about 384-414 (using HCV1 as a standard for amino acid numbering) (previously named Region V, see, for example, Ogata et al., Proc. Natl. Acad.
Sci. USA (1991) 88:3392-3396; Okamoto et ai., Virolo~v (1992) 188:331-341) appears to be the most variable region of the HCV polyprotein and is different in virtually every isolate studiE;d so far. Weiner et al., Proc. Natl. Acad. Sci.
USA
( 1992) 89:3468-3472. A number of distinct antibody-binding epitopes have been mapped to this region and in one chronically-infected patient, the emergence of an E2HV variant has been documented, suggesting that escape mutants in this E2HV
region may play an important role in the development of chronicity.
As used herein, a °'variable domain°' of a viral protein is a domain that demonstrates a consistent p;~ttern of amino acid variation between at least two HCV isolates or subpopulations. Preferably, the domain contains at least one epitope. Variable domains can vary from isolate to isolate by as little as one amino acid change. These isolates can be from the same or different HCV
groups) or subgroup(s). Variable domains can be readily identified through sequence composition among isolates, and examples of these techniques are described below. For the F~urposes of describing the present invention, variable domains will be defined with respect to the amino acid number of the polyprotein encoded by the genome of :HCVl, with the initiator methionine being designated position 1. The corresponding variable domain in another HCV isolate is determined by aligning the two isolates sequences in a manner the brings the conserved domains outside any variable domain into maximum alignment. This can be performed with any of a number of computer software packages, such as ALIGN 1.0, available from the University of Virginia, Department of Biochemistry (Attn: Dr. William R. Pearson). See Pearson et al., Proc. Natl.
Weiner et al., in FRONTIERS IN VIROLOGY: DIAGNOSIS OF HUMAN
VIRUSES BY POLYMER~rSE CHAIN REACTION TECHNOLOGY (Springer Verlag, Heidelberg, 1992). This E2 hypervariable region (E2HV) spanning amino acids about 384-414 (using HCV1 as a standard for amino acid numbering) (previously named Region V, see, for example, Ogata et al., Proc. Natl. Acad.
Sci. USA (1991) 88:3392-3396; Okamoto et ai., Virolo~v (1992) 188:331-341) appears to be the most variable region of the HCV polyprotein and is different in virtually every isolate studiE;d so far. Weiner et al., Proc. Natl. Acad. Sci.
USA
( 1992) 89:3468-3472. A number of distinct antibody-binding epitopes have been mapped to this region and in one chronically-infected patient, the emergence of an E2HV variant has been documented, suggesting that escape mutants in this E2HV
region may play an important role in the development of chronicity.
As used herein, a °'variable domain°' of a viral protein is a domain that demonstrates a consistent p;~ttern of amino acid variation between at least two HCV isolates or subpopulations. Preferably, the domain contains at least one epitope. Variable domains can vary from isolate to isolate by as little as one amino acid change. These isolates can be from the same or different HCV
groups) or subgroup(s). Variable domains can be readily identified through sequence composition among isolates, and examples of these techniques are described below. For the F~urposes of describing the present invention, variable domains will be defined with respect to the amino acid number of the polyprotein encoded by the genome of :HCVl, with the initiator methionine being designated position 1. The corresponding variable domain in another HCV isolate is determined by aligning the two isolates sequences in a manner the brings the conserved domains outside any variable domain into maximum alignment. This can be performed with any of a number of computer software packages, such as ALIGN 1.0, available from the University of Virginia, Department of Biochemistry (Attn: Dr. William R. Pearson). See Pearson et al., Proc. Natl.
Acad. Sci. USA (1988) 85:2444-2448. It is to be understood that the amino acid numbers given for a particular variable domain are somewhat subjective and a matter of choice. Thus, the beginning and end of variable domains should be understood to be approximate and to include overlapping domains or subdomains, unless otherwise indicated.
"Hypervariable domains" (HV) are variable domains exhibiting relatively high degrees of variability between isolates. In particular, the hypervariable region of HCV E2/NS 1, referred to herein as E2HV, spans amino acids 384-414.
The present invention utilizes a region within E2HV of E2/NS 1 that has a conserved motif of amino acids, referred to herein as the "SLF--G" motif for either amino acids 401 to 406 or from amino acids 401 to 407. This region was discovered by analysis of sequences of 90 isolates that encompass at least four genotypes of HCV. Antibody preparations comprised of antibodies that bind to the region with the conserved motif are useful for passive immunization against :5 HCV.
It is of course understood that the amino acids may be substituted with other molecules, for example, analogs of these amino acids, so long as the characteristics of the motif with respect to immunoreactivity are maintained.
The immunoreactivity of an antigenic determinant as compared to that of one comprised of the SLF--G motif is determinable by one of ordinary skill in the art using routine methods. For example, known methods include those used for epitope mapping, as well a;s competitive binding to antibodies that are immunologically reactive (bind) with antigenic determinants containing the motif.
As used herein, the term "polypeptide" refers to a polymer of amino acids and does not refer to a specific length of the product; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide.
This term also does not refer to or exclude post-expression modifications of the polypeptide, for example, l;lycosylations, acetylations, phosphorylations and the like. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
"Hypervariable domains" (HV) are variable domains exhibiting relatively high degrees of variability between isolates. In particular, the hypervariable region of HCV E2/NS 1, referred to herein as E2HV, spans amino acids 384-414.
The present invention utilizes a region within E2HV of E2/NS 1 that has a conserved motif of amino acids, referred to herein as the "SLF--G" motif for either amino acids 401 to 406 or from amino acids 401 to 407. This region was discovered by analysis of sequences of 90 isolates that encompass at least four genotypes of HCV. Antibody preparations comprised of antibodies that bind to the region with the conserved motif are useful for passive immunization against :5 HCV.
It is of course understood that the amino acids may be substituted with other molecules, for example, analogs of these amino acids, so long as the characteristics of the motif with respect to immunoreactivity are maintained.
The immunoreactivity of an antigenic determinant as compared to that of one comprised of the SLF--G motif is determinable by one of ordinary skill in the art using routine methods. For example, known methods include those used for epitope mapping, as well a;s competitive binding to antibodies that are immunologically reactive (bind) with antigenic determinants containing the motif.
As used herein, the term "polypeptide" refers to a polymer of amino acids and does not refer to a specific length of the product; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide.
This term also does not refer to or exclude post-expression modifications of the polypeptide, for example, l;lycosylations, acetylations, phosphorylations and the like. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
Polypeptides useful in the manufacture of the compositions of the present invention can be made recombinantly, synthetically, or in tissue culture.
Recombinant polypeptides comprised of the truncated HCV sequences or full-length HCV proteins can be made up entirely of HCV sequences (one or more epitopes, either contiguous or noncontiguous) or sequences in a fusion protein. In fusion proteins, useful heterologous sequences include sequences that provide for secretion from a recombinant host, enhance the immunological reactivity of the HCV epitope(s) or facilitate the coupling of the polypeptide to a support or a vaccine carrier. See, e.g., EPO Publ. No. 116,201; U.S. Pat. No. 4,722,840;
EPO Publ. No. 259,149; U.S. Pat. No. 4,629,783.
A significant advantage of producing the protein by recombinant DNA
techniques rather than by isolating and purifying a protein from natural sources is that equivalent quantities of the protein can be produced by using less starting material than would be required for isolating the protein from a natural source.
Producing the protein by recombinant techniques also permits the protein to be isolated in the absence of some molecules normally present in cells. Indeed, protein compositions entirely free of any trace of human protein contaminants can readily be produced because the only human protein produced by the recombinant nonhuman host is the recombinant protein at issue. Potential viral agents from natural sources and viral components pathogenic to humans are also avoided.
Polypeptides comprised of the SLF--G motif may be prepared by chemical synthesis. Methods of preparing polypeptides by chemical synthesis are known in the art. The protein may be used for producing antibodies. An "antibody" is any immunoglobulin, including antibodies and fragments thereof (including F(ab), F(ab')2, and Fv) that binds a specific epitope. The term encompasses, inter alia, polyclonal, monoclonal, single-chain, and chimeric antibodies. Examples of chimeric antibodies are discussed in U.S. Patent Nos. 4,816,397 and 4,816,567.
A polypeptide or amino acid sequence "derived from" a designated nucleic acid sequence refers to a polypeptide having an amino acid sequence identical to that of a polypeptide encoded in the sequence, or a portion thereof wherein the portion consists of at least 3-5 amino acids, and more preferably at least 8-amino acids, and even more preferably at least 11-15 amino acids, or which is im-munologically identifiable with a polypeptide encoded in the sequence. This terminology also includes a polypeptide expressed from a designated nucleic acid sequence.
The term "recombinant polynucleotide" as used herein intends a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation: ( 1 ) is not associated with all or a portion of a polynucleotide with which it is associated in nature, (2) is linked to a polynucleotide other than that to which it is linked in nature, or (3) does not occur in nature.
The term "polynucleotide" as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides.
This term refers only to the primary structure of the molecule. Thus, this term includes double- and single;-stranded DNA and RNA. It also includes known types of modifications, for example, labels which are known in the art, methylation, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e. g. , methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example proteins (including for e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide.
A "replicon" is any genetic element, e.g., a plasmid, a chromosome, a virus, ~a cosmid, etc. that behaves as an autonomous unit of polynucleotide replica-tion within a cell; i.e., capable of replication under its own control. This may include selectable markers.
A "vector" is a rep icon in which another polynucleotide segment is attached, so as to bring about the replication and/or expression of the attached segment.
r "Control sequence" refers to polynucleotide sequences which are necessary to effect the expression of coding sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding 5 site, and transcription termination sequence; in eukaryotes, generally, such control sequences include promoters and transcription termination sequence. The term "control sequences" is intended to include, at a minimum, all components whose presence is necessary for expression, and may also include additional components whose presence is advantageous, for example, leader sequences and fusion partner 10 sequences. A "promoter" is a nucleotide sequence which is comprised of consensus sequences which allow the binding of RNA polymerise to the DNA
template in a manner such that mRNA production initiates at the normal transcription initiation site for the adjacent structural gene.
"Operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.
An "open reading frame" (ORF) is a region of a polynucleotide sequence which encodes a polypeptide; this region may represent a portion of a coding sequence or a total coding sequence.
A "coding sequence" is a polynucleotide sequence which is translated into a polypeptide, usually via mRNA, when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5'-terminus and a translation stop codon at the 3'-terminus. A coding sequence can include, but is not limited to, cDNA, and recombinant polynucleotide sequences.
"PCR" refers to the technique of polymerise chain reaction as described in Saiki et al., Nature (1986) 324:163; Scharf et al., Science (1986) 233:1076-1078;
U.S. Patent No. 4,683,195; and U.S. Patent No. 4,683.202.
Recombinant polypeptides comprised of the truncated HCV sequences or full-length HCV proteins can be made up entirely of HCV sequences (one or more epitopes, either contiguous or noncontiguous) or sequences in a fusion protein. In fusion proteins, useful heterologous sequences include sequences that provide for secretion from a recombinant host, enhance the immunological reactivity of the HCV epitope(s) or facilitate the coupling of the polypeptide to a support or a vaccine carrier. See, e.g., EPO Publ. No. 116,201; U.S. Pat. No. 4,722,840;
EPO Publ. No. 259,149; U.S. Pat. No. 4,629,783.
A significant advantage of producing the protein by recombinant DNA
techniques rather than by isolating and purifying a protein from natural sources is that equivalent quantities of the protein can be produced by using less starting material than would be required for isolating the protein from a natural source.
Producing the protein by recombinant techniques also permits the protein to be isolated in the absence of some molecules normally present in cells. Indeed, protein compositions entirely free of any trace of human protein contaminants can readily be produced because the only human protein produced by the recombinant nonhuman host is the recombinant protein at issue. Potential viral agents from natural sources and viral components pathogenic to humans are also avoided.
Polypeptides comprised of the SLF--G motif may be prepared by chemical synthesis. Methods of preparing polypeptides by chemical synthesis are known in the art. The protein may be used for producing antibodies. An "antibody" is any immunoglobulin, including antibodies and fragments thereof (including F(ab), F(ab')2, and Fv) that binds a specific epitope. The term encompasses, inter alia, polyclonal, monoclonal, single-chain, and chimeric antibodies. Examples of chimeric antibodies are discussed in U.S. Patent Nos. 4,816,397 and 4,816,567.
A polypeptide or amino acid sequence "derived from" a designated nucleic acid sequence refers to a polypeptide having an amino acid sequence identical to that of a polypeptide encoded in the sequence, or a portion thereof wherein the portion consists of at least 3-5 amino acids, and more preferably at least 8-amino acids, and even more preferably at least 11-15 amino acids, or which is im-munologically identifiable with a polypeptide encoded in the sequence. This terminology also includes a polypeptide expressed from a designated nucleic acid sequence.
The term "recombinant polynucleotide" as used herein intends a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation: ( 1 ) is not associated with all or a portion of a polynucleotide with which it is associated in nature, (2) is linked to a polynucleotide other than that to which it is linked in nature, or (3) does not occur in nature.
The term "polynucleotide" as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides.
This term refers only to the primary structure of the molecule. Thus, this term includes double- and single;-stranded DNA and RNA. It also includes known types of modifications, for example, labels which are known in the art, methylation, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e. g. , methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example proteins (including for e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide.
A "replicon" is any genetic element, e.g., a plasmid, a chromosome, a virus, ~a cosmid, etc. that behaves as an autonomous unit of polynucleotide replica-tion within a cell; i.e., capable of replication under its own control. This may include selectable markers.
A "vector" is a rep icon in which another polynucleotide segment is attached, so as to bring about the replication and/or expression of the attached segment.
r "Control sequence" refers to polynucleotide sequences which are necessary to effect the expression of coding sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding 5 site, and transcription termination sequence; in eukaryotes, generally, such control sequences include promoters and transcription termination sequence. The term "control sequences" is intended to include, at a minimum, all components whose presence is necessary for expression, and may also include additional components whose presence is advantageous, for example, leader sequences and fusion partner 10 sequences. A "promoter" is a nucleotide sequence which is comprised of consensus sequences which allow the binding of RNA polymerise to the DNA
template in a manner such that mRNA production initiates at the normal transcription initiation site for the adjacent structural gene.
"Operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.
An "open reading frame" (ORF) is a region of a polynucleotide sequence which encodes a polypeptide; this region may represent a portion of a coding sequence or a total coding sequence.
A "coding sequence" is a polynucleotide sequence which is translated into a polypeptide, usually via mRNA, when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5'-terminus and a translation stop codon at the 3'-terminus. A coding sequence can include, but is not limited to, cDNA, and recombinant polynucleotide sequences.
"PCR" refers to the technique of polymerise chain reaction as described in Saiki et al., Nature (1986) 324:163; Scharf et al., Science (1986) 233:1076-1078;
U.S. Patent No. 4,683,195; and U.S. Patent No. 4,683.202.
As used herein, x is "heterologous" with respect to y if x is not naturally associated with y in the identical manner; i.e., x is not associated with y in nature or x is not associated with y in the same manner as is found in nature.
"Homology" refers to the degree of similarity between x and y. Homology between two polynucleotidc: sequences can be determined by techniques known in the art. For example, it cam be determined by a direct comparison of the sequence information of the polynuc:~(eotide. Alternatively, homology can be determined by hybridization of the polynucleotides under conditions which form stable duplexes between homologous regions (for example, those which would be used prior to S1 digestion), followed by digestion with single-stranded specific nuclease(s), fol-Iowed by size determination of the digested fragments.
"Recombinant host cells" "host cells " "cells " "cell cultures " and other > > > , such terms denote, for example, microorganisms, insect cells, and mammalian cells, that can be, or have been, used as recipients for recombinant vector or other transfer DNA, and include the progeny of the original cell which has been transformed. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA
complement as the original parent, due to natural, accidental, or deliberate mutation. Examples for mammalian host cells include Chinese hamster ovary (CHO) and monkey kidney (COS) cells.
Specifically, as used herein, "cell line" refers to a population of cells capable of continuous or prolonged growth and division in vitro. Often, cell lines are clonal populations derived from a single progenitor cell. It is further known in the art that spontaneous or induced changes can occur in karyotype during storage or transfer of such clonal populations. Therefore, cells derived from the cell line referred to may not be precisely identical to the ancestral cells or cultures, and the cell line referred to includes such variants. The term "cell lines" also includes immortalized cells. Preferably, cell lines include nonhybrid cell lines or hybridomas to only two cell types.
--a used herein, the term "microorganism" includes prokaryotic and eukaryotic microbial species such as bacteria and fungi, the latter including yeast and filamentous fungi.
WO 94/26306 PCTlUS94l04853 "Transformation" refers to the insertion of an exogenous polynucleotide into a host cell, irrespective of the method used for the insertion, for example, direct uptake, transduction, f-mating or electroporation. The exogenous polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host genome.
By "genomic" is meant a collection or library of DNA molecules which are derived from restriction fragments that have been cloned in vectors. This may include all or part of the genetic material of an organism.
By "cDNA" is meant a complimentary mRNA sequence that hybridizes to a complimentary strand of mRNA.
By "purified" and "isolated" is meant, when refernng to a polypeptide or nucleotide sequence, that the indicated molecule is present in the substantial absence of other biological macromolecules of the same type. The term "purified"
as used herein preferably means at least 75 % by weight, more preferably at least 85 % by weight, more preferably still at least 95 % by weight, and most preferably at least 98 % by weight, of biological macromolecules of the same type present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 1000, can be present).
As used herein, "epitope" is a single antigenic determinant which has a structure complementary to the recognition site on a lymphocyte receptor or an antibody. Functionally, it is determined by the ability of an antigen to bind to an antibody in a standard assay. Generally, an epitope comprises at least 3 to 5 amino acids. Sometimes, epitopes can be larger, e.g., 6, 7, 8, 9, or 10 amino acids.
An epitope or antigenic determinant is the equivalent of another epitope or antigenic determinant in a designated polypeptide when it cross-reacts with antibodies which bind immunologically to the epitope or antigenic determinant in the designated polypeptide. Often, these are one or more amino acids within an epitope that are not critical for antibody binding and are thus capable of substitution or even deletion. Although linear epitopes are usually short, contiguous sequences (subject to some change), conformational epitopes can be comprised of a few amino acids widely spaced within the linear amino acid O 94!26306 PCTlUS94l04853 sequence, but brought within close proximity due to folding or other secondary or tertiary structural features of the protein.
An "antigen" is a polypeptide containing one or more antigenic determinants.
"Immunogenic" means the ability to elicit a cellular and/or humoral immune response. An immunogenic response may be elicited by immunogenic polypeptides alone, or may require the presence of a carrier in the presence or absence of an adjuvant.
"Immunoreactive" refers to ( 1 ) the ability to bind immunologically to an antibody and/or to a lymphocyte antigen receptor or (2) the ability to be immunogenic.
The amino acid s~uence comprising the HCV epitope may be linked to another polypeptide (e.g., a carrier protein), either by covalent attachment or by expressing a fused polynuc;leotide to form a fusion protein. If desired, one may insert or attach multiple repeats of the epitope, and/or incorporate a variety of epitopes. The carrier protein may be derived from any source, but will generally be a relatively large, immunogenic protein such as BSA, KLH, or the like. If desired, one may employ a substantially full-length HCV protein as the carrier, multiplying the number of immunogenic epitopes. Alternatively, the amino acid sequence from the HCV epitope may be linked at the amino terminus and/or carboxy terminus to a non-HCV amino acid sequence, thus the polypeptide would be a fusion polypeptide. Analogous types of polypeptides may be constructed using epitopes from other designated viral proteins.
An "individual" refers to a vertebrate, particularly a member of a mammalian species, and includes but is not limited to rodents (e.g., mice, rats, hamsters, guinea pigs), rabbits, goats, pigs, cattle, sheep, and primates (e.g., chimpanzees, African Green Monkeys, baboons, orangutans, and humans).
As used herein, "treatment" refers to any of (i) the prevention of infection or reinfection, as in a traditional vaccine, (ii) the reduction or elimination of one or more symptoms associated with an HCV infected state, and (iii) the substantial or complete elimination of the virus. Treatment may be effected prophylactically (prior to infection) or therapeutically (following infection).
"Homology" refers to the degree of similarity between x and y. Homology between two polynucleotidc: sequences can be determined by techniques known in the art. For example, it cam be determined by a direct comparison of the sequence information of the polynuc:~(eotide. Alternatively, homology can be determined by hybridization of the polynucleotides under conditions which form stable duplexes between homologous regions (for example, those which would be used prior to S1 digestion), followed by digestion with single-stranded specific nuclease(s), fol-Iowed by size determination of the digested fragments.
"Recombinant host cells" "host cells " "cells " "cell cultures " and other > > > , such terms denote, for example, microorganisms, insect cells, and mammalian cells, that can be, or have been, used as recipients for recombinant vector or other transfer DNA, and include the progeny of the original cell which has been transformed. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA
complement as the original parent, due to natural, accidental, or deliberate mutation. Examples for mammalian host cells include Chinese hamster ovary (CHO) and monkey kidney (COS) cells.
Specifically, as used herein, "cell line" refers to a population of cells capable of continuous or prolonged growth and division in vitro. Often, cell lines are clonal populations derived from a single progenitor cell. It is further known in the art that spontaneous or induced changes can occur in karyotype during storage or transfer of such clonal populations. Therefore, cells derived from the cell line referred to may not be precisely identical to the ancestral cells or cultures, and the cell line referred to includes such variants. The term "cell lines" also includes immortalized cells. Preferably, cell lines include nonhybrid cell lines or hybridomas to only two cell types.
--a used herein, the term "microorganism" includes prokaryotic and eukaryotic microbial species such as bacteria and fungi, the latter including yeast and filamentous fungi.
WO 94/26306 PCTlUS94l04853 "Transformation" refers to the insertion of an exogenous polynucleotide into a host cell, irrespective of the method used for the insertion, for example, direct uptake, transduction, f-mating or electroporation. The exogenous polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host genome.
By "genomic" is meant a collection or library of DNA molecules which are derived from restriction fragments that have been cloned in vectors. This may include all or part of the genetic material of an organism.
By "cDNA" is meant a complimentary mRNA sequence that hybridizes to a complimentary strand of mRNA.
By "purified" and "isolated" is meant, when refernng to a polypeptide or nucleotide sequence, that the indicated molecule is present in the substantial absence of other biological macromolecules of the same type. The term "purified"
as used herein preferably means at least 75 % by weight, more preferably at least 85 % by weight, more preferably still at least 95 % by weight, and most preferably at least 98 % by weight, of biological macromolecules of the same type present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 1000, can be present).
As used herein, "epitope" is a single antigenic determinant which has a structure complementary to the recognition site on a lymphocyte receptor or an antibody. Functionally, it is determined by the ability of an antigen to bind to an antibody in a standard assay. Generally, an epitope comprises at least 3 to 5 amino acids. Sometimes, epitopes can be larger, e.g., 6, 7, 8, 9, or 10 amino acids.
An epitope or antigenic determinant is the equivalent of another epitope or antigenic determinant in a designated polypeptide when it cross-reacts with antibodies which bind immunologically to the epitope or antigenic determinant in the designated polypeptide. Often, these are one or more amino acids within an epitope that are not critical for antibody binding and are thus capable of substitution or even deletion. Although linear epitopes are usually short, contiguous sequences (subject to some change), conformational epitopes can be comprised of a few amino acids widely spaced within the linear amino acid O 94!26306 PCTlUS94l04853 sequence, but brought within close proximity due to folding or other secondary or tertiary structural features of the protein.
An "antigen" is a polypeptide containing one or more antigenic determinants.
"Immunogenic" means the ability to elicit a cellular and/or humoral immune response. An immunogenic response may be elicited by immunogenic polypeptides alone, or may require the presence of a carrier in the presence or absence of an adjuvant.
"Immunoreactive" refers to ( 1 ) the ability to bind immunologically to an antibody and/or to a lymphocyte antigen receptor or (2) the ability to be immunogenic.
The amino acid s~uence comprising the HCV epitope may be linked to another polypeptide (e.g., a carrier protein), either by covalent attachment or by expressing a fused polynuc;leotide to form a fusion protein. If desired, one may insert or attach multiple repeats of the epitope, and/or incorporate a variety of epitopes. The carrier protein may be derived from any source, but will generally be a relatively large, immunogenic protein such as BSA, KLH, or the like. If desired, one may employ a substantially full-length HCV protein as the carrier, multiplying the number of immunogenic epitopes. Alternatively, the amino acid sequence from the HCV epitope may be linked at the amino terminus and/or carboxy terminus to a non-HCV amino acid sequence, thus the polypeptide would be a fusion polypeptide. Analogous types of polypeptides may be constructed using epitopes from other designated viral proteins.
An "individual" refers to a vertebrate, particularly a member of a mammalian species, and includes but is not limited to rodents (e.g., mice, rats, hamsters, guinea pigs), rabbits, goats, pigs, cattle, sheep, and primates (e.g., chimpanzees, African Green Monkeys, baboons, orangutans, and humans).
As used herein, "treatment" refers to any of (i) the prevention of infection or reinfection, as in a traditional vaccine, (ii) the reduction or elimination of one or more symptoms associated with an HCV infected state, and (iii) the substantial or complete elimination of the virus. Treatment may be effected prophylactically (prior to infection) or therapeutically (following infection).
As used herein; a "biological sample" refers to a sample of tissue or fluid isolated from an individual, including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs, biopsies and also samples of in vitro cell culture constituents (including but not limited to conditioned medium resulting from the growth of cells in cell culture medium, e.g., Mab producing myeloma cells, recombinant cells, and cell components) .
An "immune response" to a composition or vaccine is the development in the host of a cellular and/or antibody-mediated immune response to the intracellular infectious agent that encodes the target antigen. Usually, such a response comprises the individual producing cytotoxic T cells and/or B cells and/or a variety of classes of T cells directed specifically to antigen presenting cells expressing the target antigen.
B. Expression Systems The availability of DNA sequences encoding the polypeptides of this invention permits the construction of expression vectors encoding these polypeptides. The DNA encoding the desired polypeptide, whether in fused or mature form, and whether or not containing a signal sequence to permit secretion, may be ligated into expression vectors suitable for any convenient host. Both eukaryotic and prokaryotic host systems are presently used in forming recombinant polypeptides, and a summary of some of the more common control systems and host cell lines is given below. The polypeptide is then isolated from lysed cells or from the culture medium and purified to the extent needed for its intended use.
Purification may be by techniques known in the art, for example, differential extraction, salt fractionation, chromatography on ion exchange resins, affinity chromatography, centrifugation, and the like. See, for example, Methods in Enzymology {Academic Press) for a variety of methods for purifying proteins.
Such polypeptides can be used as diagnostics, or those which give rise to neutral-izing antibodies may be formulated into vaccines. Antibodies raised against these polypeptides can also be used as diagnostics, or for passive immunotherapy.
O 94!26306 PCTIUS94/04853 Both prokaryotic and eukaryotic host cells may be used for expression of desired coding sequences when appropriate control sequences which are compatible with the designated host are used. Methods for such expression are known in the art. Amont; prokaryotic hosts, E. coli is most frequently used.
5 Expression control sequences for prokaryotes include promoters, optionally containing operator portions, and ribosome binding sites. Transfer vectors com-patible with prokaryotic hosts are commonly derived from, for example, pBR322, a plasmid containing operons conferring ampicillin and tetracycline resistance, and the various pUC vectors, which also contain sequences conferring antibiotic resist-10 ance markers. These markers may be used to obtain successful transformants by selection. Commonly used prokaryotic control sequences include the B-lactamase (penicillinase) and lactose promoter systems (Chang et al. , Nature ( 1977) 198:1056), the tryptophan (tip) promoter system (Goeddel et al. , Nucleic Acids Res. (1980) 8:4057) and t:he lambda-derived PL promoter and N gene ribosome 15 binding site (Shimatake et al., Nature (1981) 292:128) and the hybrid tac promoter (De Boer et al., Proc. Natl. Acad. Sci. USA (1983) 292:128) derived from sequences of the try and lac U'VS promoters. The foregoing systems are particularly compatible wiith E. coli; if desired, other prokaryotic hosts such as strains of Bacillus or Pseudomonas may be used, with corresponding control sequences.
Eukaryotic hosts include yeast and mammalian cells in culture systems.
Saccharom,~ cerevisiae and Saccharom,~ carlsber ensis are the most commonly used yeast hosos, and are convenient fungal hosts. Yeast compatible vectors carry markers which permit selection of successful transformants by con-fernng prototrophy to aux:otrophic mutants or resistance to heavy metals on wild-type strains. Yeast compatible vectors may employ the 2~, origin of replication (Broach et al., Meth. Enz_ (1983) 101:307), the combination of CEN3 and ARSI
or other means for assuring replication, such as sequences which will result in in-corporation of an appropriate fragment into the host cell genome. Control sequences for yeast vectors are known in the art and include promoters for the synthesis of glycolytic en2:ymes (Hess et al., N. Adv. Enzyme Rep (1968) 7:149;
Holland et al., Biochemistry (1978) .17:4900), including the promoter for 3-16 ~ ~ 62557 phosphoglycerate kinase (Fiitzeman, J. Biol. Chem. (1980) 55:2073).
Terminators may also be included, such as those derived from the enolase gene (Holland, J. Biol. Chem. (1981) 2~: 1385). Particularly useful contml systems are those which comprise the glyceraldehyde-3 phosphate dehydrogenase (GAPDH) promoter or al~"ohol dehydrogenase (ADH) regulatable promoter, terminators also derived from GAPDH, and if secretion is desired, leader sequence from yeast a-factor. In addition, the transcriptional regulatory region and the transcriptional initiation region which are operably linked may be such that they are not naturally associated in the wild-type organism. These systems are described in detail in EPO 120,551, published October 3, 1984; EPO 116,201, published August 22, 19&4; and EPO 164,556, published December 18, 1985, all of which are assigned to the herein- assignee"
Mammalian cell lines available as hosts for expression are known in the art ,and include many immortalized cell lines available from the American Type Culture Collection (ATCC;), including HeLa cells, Chinese hamster ovary (CHO) cells, baby hamster kidney (BHI~ cells, and a number of other cell lines.
Suitable promoters for mammalian cells are also known in the art and include viral promoters such as that from Simian Virus 40 (SV40) (Hers, Nature (1978) 27:113), Rous sarcoma virus (RSV), adenovirus (ADV), and bovine papiiloma virus (BPV). Mammalian cells may also require terminator sequences and poly-A
addition sequences; enhancer sequences which increase expression may also be included, and sequences which cause amplification of the gene may also be desirable. These sequenG~s are known in the art.
Vectors suitable for replication in mammalian cells are known in the art, and may include viral replicons, or sequences which insure integration of the appropriate sequences encoding NANBV epitopes into the host genome.
A vector which is used to express foreign DNA, and which may be used in vaccine preparation is Vac:cinia virus. In this case the heterologous DNA is inserted into the Vaccinia genome. Techniques for the insertion of foreign DNA
into the vaccinia virus genome are known in the art, and utilize, for example, homologous recombination. The insertion of the heterologous DNA is generally ,:
.'O 94/26306 ~'~ PCT/US94104853 into a gene which is non-essential in nature, for example, the thymidine kinase gene (tk), which also provides a selectable. marker. Plasmid vectors that greatly facilitate the construction of recombinant viruses have been described (see, for example, Mackett et al., J. Virol. (1984) 49:857: Chakrabarti et al., Mol.
Cell Biol. (1985) 5:3403; Moss. (1987) in GENE TRANSFER VECTORS FOR
MAMMALIAN CELLS (lvliller and Calos, eds., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), p. 10.). Expression of the HCV polypeptide then occurs in cells or individuals which are immunized with the live recombinant vaccinia virus.
Other systems for expression of eukaryotic or viral genomes include insect cells and vectors suitable for use in these cells. These systems are known in the art, and include, for example, insect expression transfer vectors derived from the baculovirus Autographs ca.lifornica nuclear polyhedrosis virus (AcNPV), which is a helper-independent, wall expression vector for use in Spodoptera frugiperda cells in culture, for example. Expression vectors derived from this system usually use the strong viral polyhe~drin gene promoter to drive expression of heterologous genes. Currently the most commonly used transfer vector for introducing foreign genes into AcNPV is pAc:373 (Fig. 70). Many other vectors, known to those of skill in the art, have also been designed for improved expression. These include, for example, pVL985 (which alters the polyhedrin start codon from ATG to ATT, and which introduces a BamHI cloning site 32 basepairs downstream from the ATT; See Luckow and Summers, ViroloQV (1989) 17:31.).
Methods for the introduction of heterologous DNA into the desired site in the baculovirus virus are known in the art. (See Summer and Smith, Texas Agricultural Experiment Station Bulletin No. 1555; ; Smith et al., Mol. & Cell Biol. (1983) 3:2156-2165.; and Luckow and Summers (1989)). For example, the insertion can be into a gene such as the polyhedrin gene, by homologous recom-bination; insertion can also be into a restriction enzyme site engineered into the desired baculovirus gene.
C. Vaccine Treatment of HCV
In one embodimenvt of the invention, the immunogenic compositions comprised of a polypeptide having a region that binds an antibody directed to an WO 94126306 PCTIUS94l04853 ,.
An "immune response" to a composition or vaccine is the development in the host of a cellular and/or antibody-mediated immune response to the intracellular infectious agent that encodes the target antigen. Usually, such a response comprises the individual producing cytotoxic T cells and/or B cells and/or a variety of classes of T cells directed specifically to antigen presenting cells expressing the target antigen.
B. Expression Systems The availability of DNA sequences encoding the polypeptides of this invention permits the construction of expression vectors encoding these polypeptides. The DNA encoding the desired polypeptide, whether in fused or mature form, and whether or not containing a signal sequence to permit secretion, may be ligated into expression vectors suitable for any convenient host. Both eukaryotic and prokaryotic host systems are presently used in forming recombinant polypeptides, and a summary of some of the more common control systems and host cell lines is given below. The polypeptide is then isolated from lysed cells or from the culture medium and purified to the extent needed for its intended use.
Purification may be by techniques known in the art, for example, differential extraction, salt fractionation, chromatography on ion exchange resins, affinity chromatography, centrifugation, and the like. See, for example, Methods in Enzymology {Academic Press) for a variety of methods for purifying proteins.
Such polypeptides can be used as diagnostics, or those which give rise to neutral-izing antibodies may be formulated into vaccines. Antibodies raised against these polypeptides can also be used as diagnostics, or for passive immunotherapy.
O 94!26306 PCTIUS94/04853 Both prokaryotic and eukaryotic host cells may be used for expression of desired coding sequences when appropriate control sequences which are compatible with the designated host are used. Methods for such expression are known in the art. Amont; prokaryotic hosts, E. coli is most frequently used.
5 Expression control sequences for prokaryotes include promoters, optionally containing operator portions, and ribosome binding sites. Transfer vectors com-patible with prokaryotic hosts are commonly derived from, for example, pBR322, a plasmid containing operons conferring ampicillin and tetracycline resistance, and the various pUC vectors, which also contain sequences conferring antibiotic resist-10 ance markers. These markers may be used to obtain successful transformants by selection. Commonly used prokaryotic control sequences include the B-lactamase (penicillinase) and lactose promoter systems (Chang et al. , Nature ( 1977) 198:1056), the tryptophan (tip) promoter system (Goeddel et al. , Nucleic Acids Res. (1980) 8:4057) and t:he lambda-derived PL promoter and N gene ribosome 15 binding site (Shimatake et al., Nature (1981) 292:128) and the hybrid tac promoter (De Boer et al., Proc. Natl. Acad. Sci. USA (1983) 292:128) derived from sequences of the try and lac U'VS promoters. The foregoing systems are particularly compatible wiith E. coli; if desired, other prokaryotic hosts such as strains of Bacillus or Pseudomonas may be used, with corresponding control sequences.
Eukaryotic hosts include yeast and mammalian cells in culture systems.
Saccharom,~ cerevisiae and Saccharom,~ carlsber ensis are the most commonly used yeast hosos, and are convenient fungal hosts. Yeast compatible vectors carry markers which permit selection of successful transformants by con-fernng prototrophy to aux:otrophic mutants or resistance to heavy metals on wild-type strains. Yeast compatible vectors may employ the 2~, origin of replication (Broach et al., Meth. Enz_ (1983) 101:307), the combination of CEN3 and ARSI
or other means for assuring replication, such as sequences which will result in in-corporation of an appropriate fragment into the host cell genome. Control sequences for yeast vectors are known in the art and include promoters for the synthesis of glycolytic en2:ymes (Hess et al., N. Adv. Enzyme Rep (1968) 7:149;
Holland et al., Biochemistry (1978) .17:4900), including the promoter for 3-16 ~ ~ 62557 phosphoglycerate kinase (Fiitzeman, J. Biol. Chem. (1980) 55:2073).
Terminators may also be included, such as those derived from the enolase gene (Holland, J. Biol. Chem. (1981) 2~: 1385). Particularly useful contml systems are those which comprise the glyceraldehyde-3 phosphate dehydrogenase (GAPDH) promoter or al~"ohol dehydrogenase (ADH) regulatable promoter, terminators also derived from GAPDH, and if secretion is desired, leader sequence from yeast a-factor. In addition, the transcriptional regulatory region and the transcriptional initiation region which are operably linked may be such that they are not naturally associated in the wild-type organism. These systems are described in detail in EPO 120,551, published October 3, 1984; EPO 116,201, published August 22, 19&4; and EPO 164,556, published December 18, 1985, all of which are assigned to the herein- assignee"
Mammalian cell lines available as hosts for expression are known in the art ,and include many immortalized cell lines available from the American Type Culture Collection (ATCC;), including HeLa cells, Chinese hamster ovary (CHO) cells, baby hamster kidney (BHI~ cells, and a number of other cell lines.
Suitable promoters for mammalian cells are also known in the art and include viral promoters such as that from Simian Virus 40 (SV40) (Hers, Nature (1978) 27:113), Rous sarcoma virus (RSV), adenovirus (ADV), and bovine papiiloma virus (BPV). Mammalian cells may also require terminator sequences and poly-A
addition sequences; enhancer sequences which increase expression may also be included, and sequences which cause amplification of the gene may also be desirable. These sequenG~s are known in the art.
Vectors suitable for replication in mammalian cells are known in the art, and may include viral replicons, or sequences which insure integration of the appropriate sequences encoding NANBV epitopes into the host genome.
A vector which is used to express foreign DNA, and which may be used in vaccine preparation is Vac:cinia virus. In this case the heterologous DNA is inserted into the Vaccinia genome. Techniques for the insertion of foreign DNA
into the vaccinia virus genome are known in the art, and utilize, for example, homologous recombination. The insertion of the heterologous DNA is generally ,:
.'O 94/26306 ~'~ PCT/US94104853 into a gene which is non-essential in nature, for example, the thymidine kinase gene (tk), which also provides a selectable. marker. Plasmid vectors that greatly facilitate the construction of recombinant viruses have been described (see, for example, Mackett et al., J. Virol. (1984) 49:857: Chakrabarti et al., Mol.
Cell Biol. (1985) 5:3403; Moss. (1987) in GENE TRANSFER VECTORS FOR
MAMMALIAN CELLS (lvliller and Calos, eds., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), p. 10.). Expression of the HCV polypeptide then occurs in cells or individuals which are immunized with the live recombinant vaccinia virus.
Other systems for expression of eukaryotic or viral genomes include insect cells and vectors suitable for use in these cells. These systems are known in the art, and include, for example, insect expression transfer vectors derived from the baculovirus Autographs ca.lifornica nuclear polyhedrosis virus (AcNPV), which is a helper-independent, wall expression vector for use in Spodoptera frugiperda cells in culture, for example. Expression vectors derived from this system usually use the strong viral polyhe~drin gene promoter to drive expression of heterologous genes. Currently the most commonly used transfer vector for introducing foreign genes into AcNPV is pAc:373 (Fig. 70). Many other vectors, known to those of skill in the art, have also been designed for improved expression. These include, for example, pVL985 (which alters the polyhedrin start codon from ATG to ATT, and which introduces a BamHI cloning site 32 basepairs downstream from the ATT; See Luckow and Summers, ViroloQV (1989) 17:31.).
Methods for the introduction of heterologous DNA into the desired site in the baculovirus virus are known in the art. (See Summer and Smith, Texas Agricultural Experiment Station Bulletin No. 1555; ; Smith et al., Mol. & Cell Biol. (1983) 3:2156-2165.; and Luckow and Summers (1989)). For example, the insertion can be into a gene such as the polyhedrin gene, by homologous recom-bination; insertion can also be into a restriction enzyme site engineered into the desired baculovirus gene.
C. Vaccine Treatment of HCV
In one embodimenvt of the invention, the immunogenic compositions comprised of a polypeptide having a region that binds an antibody directed to an WO 94126306 PCTIUS94l04853 ,.
antigenic determinant containing the SLF--G motif is used for vaccine applications to stimulate immune responsiveness to the HCV antigenic determinant{s) containing the motif. Preferably, the polypeptides do not contain the specific E2HV sequences disclosed in PCT Publ. No. W093/016126; Weiner et al., Virolo~v (1991) 180:842-48; Weiner et al., Proc. Natl. Acad. Sci. USA (1992) 89:3468-72; Weiner et al. (1992), Vaccines 92:303-08, Cold Spring Harbor Laboratory.
Preliminary evidence suggests that the hypervariable domains) of E2/NS 1 may be responsible for escape mutants, leading to chronic HCV infections.
However, a conserved region within the hypervariable region is suggestive that the conserved region has an important function and plays an essential role in virus binding andlor entry into and/or replication in the host cell. In virus binding it is contemplated that the binding may be to the cell and/or to another molecule which facilitates virus binding and/or entry and/or replication. The examples presented infra. are suggestive that virus binding to transthyretin and/or to thyroid binding globulin (TBG) are involved in the infective process. Thus, increasing an immune response to antigenic determinants containing the conserved SLF--G sequence may lead not only to protection against and/or alleviation of HCV infection, but also to a reduction in chronicity of HCV infection. In addition, the conserved region is also suggestive that the vaccines comprised of the immununoreactive polypeptides having a region with the SLF--G motif may be cross-reactive.
In preferred applications for vaccines, the polypeptide compositions described herein are combined with other HCV subunit antigens, for example, those described in PCT Publ. No. W092/08734. In cases where the composition is to be used for treatment of HCV, it is desirable that the composition be immunogenic. In instances wherein the synthesized polypeptide is correctly configured so as to provide the correct epitope, but is too small to be immunogenic, the polypeptide may be linked to a suitable Garner. A number of techniques for obtaining such linkage are known in the art, including the formation of disulfide linkages using N-succinimidyl-3-(2-pyridyl-thio)propionate (SPDP) and succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) (if the peptide lacks a sulfhydryl group, this can be provided by addition of a cysteine !O 94126306 residue. ) These reagents create a disulfide linkage between themselves and peptide cysteine resides on one protein and an amide linkage through the e-amino on a lysine, or other free amino group in other amino acids. A variety of such disulfide/amide-forming agents are known. See, for example, Immun. Rev.
( 1982) 62:185. Other bifunctional coupling agents for a thioether rather than a disulfide linkage. Many of these thio-ether-forming agents are commercially available and include reactive esters of 6-maleimidocaproic acid, 2-bromoacetic acid, 2-iodoacetic acid, 4-((~T-maleimido-methyl)cyclohexane-1-carboxylic acid, and the like. The carboxyl groups can be activated by combining them with succinimide or 1-hydroxyl-2-nitro-4-sulfonic acid, sodium salt. Additional methods of coupling antigens employ the rotavirus/°'binding peptide"
system described in EPO Publ. No. 259,149. The foregoing list is not meant to be exhaustive, and modifications of the named compounds can clearly be used.
Any carrier may be used which does not itself induce the production of antibodies harmful to the host. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins; polysaccharides such as latex functionalized sepharose, a;garose, cellulose, cellulose beads and the like;
polymeric amino acids, such as polyglutamic acid, polylysine, and the like;
amino acid copolymers; and inactive virus particles (see infra.). Especially useful protein substrates are serum albumins, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, crvalbumin, tetanus toxoid, and other proteins well known to those of skill in t:he art.
The immunogenicity of the antigens comprised of the SLF--G motif may also be enhanced by prepaung them in eukaryotic systems fused with or assembled with particle-forming proteins such as, for example, that associated with hepatitis B surface antigen. See, e.;~., U.S. Patent No. 4,722,840. These constructs may also be expressed in mammalian cells such as CHO cells using an SV40-dihydrofolate reductase vector (Michelle et al. ( 1984), INTERNATIONAL
SYMPOSIUM ON VIRAL HEPATITIS).
In addition, portions of the particle-forming protein coding sequence may be replaced with codons encoding the SLF--G epitope from an HCV hypervariable domain. In this replacement, regions which are not required to mediate the 20 2 ~ 6~55~
aggregation of the units to form immunogenic particles in yeast yr mammals can be deleted, thus eliminating additional HBV antigenic sites from competition with the HCV epitope(s).
These vaccines ma~,y either be prophylactic (to prevent infection) or therapeutic (to treat disea:~e after infection).
Such vaccines comprise antigen or antigens, usually in combination with "pharmaceutically acceptable carriers," which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles. Such carriers are well known to those of ordinary skill iin the aut. Additionally, these carriers may function as immunostimulating agents ("adjuvants"). Furthermore, the antigen may be conjugated to a bacterial toxoid, such as a toxoid from diphtheria, tetanus, cholera, -~I , etc. pathogens.
Preferred adjuvant;s to enhance effectiveness of the composition include, but are not limited to: (1) aauminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aaunninum sulfate, etc; (2) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides (see below) or bacterial cell wall components), such as for example (a) MF59 (PCT Publ. No. WO 90114837), containing 5 % Squalene* 0.5 % Tween*80, and 0.5 ~o Span 85'"(optior.~ally containing various amounts of MTP-PE (see below), although not required) formulated into submicmn particles ,using a microfluidizer such as Model llOY microfluidizer (Microfluidics, Newton, MA), (b) SAF, containing 10'~ Squalane;k0.4 ~ Tweeri 80, 5 % pluronic-blocked polymer Ll 21, and thr-MDP (see below) either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) R.ibrT'"
adjuvant system (RAS), (Ribi Imm~unochem, Hamilton, MT) containing 2 % Squalen~, 0.2 Tween*$0, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate ('TDM), and cell wall skeleton (CWS), prederably MPL + CWS (DetoxT"'); (3) saponin adjuvants, * Tween - trade-mark * Span 85 - trade-mark *Squalane - trade-mark * Squalene - trade-mark O 94/26306 ~ ~ PCTlUS94104853 such as StimulonTM (Cambridge Bioscience, Worcester, MA) may be used or particles generated therefrom such as ISCOMs (immunostimulating complexes);
(4) Complete Freunds Adjuvant (CFA) and Incomplete Freunds Adjuvant (IFA);
(5) cytokines, such as interleukins (IL-l, IL-2, etc.), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc; and (6) other substances that act as immunostimulating agents to enhance the effectiveness of the composition. Alum and N1F59 are preferred.
As mentioned above, muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-( 1 '-2'-dipalinitoyl-sn-glycero-3-hydroxyphosphoryioxy)-ethylamine (MTP-PE), etc.
The immunogenic compositions (e.g., the antigen, pharmaceutically acceptable carrier, and adjuvant) typically will contain diluents, such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.
Typically, the imm unogenic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation also may be emulsified or encapsulated in liposomes for enhanced adjuvant effect, as discussf:d above under pharmaceutically acceptable carriers.
Immunogenic compositions used as vaccines comprise an immunologically effective amount of the antigenic polypeptides, as well as any other of the above-mentioned components, as needed. "Immunologically effective amount" means that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment. This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated (e. g. , nonhuman primate, primate, etc. ), the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the WO 94126306 PCTlUS94104853 medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
The immunogenic compositions are conventionally administered parenterally, e.g., by injection, either subcutaneously or intramuscularly.
Additional formulations suitable for other modes of administration include oral and pulinonary formulations, suppositories, and transdermal applications. Dosage treatment may be a single dose schedule or a multiple dose schedule. The vaccine may be administered in conjunction with other immunoregulatory agents.
In addition to the above, it is also possible to prepare live vaccines of attenuated microorganisms which express recombinant polypeptides comprised of a region with the SLF--G motif. Suitable attenuated microorganisms are known in the art and include, for example, viruses (e.g., vaccinia virus) as well as bacteria.
In addition, the vaccine containing the polypeptide with an antigenic determinant comprised of the conserved motif SLF--G may be administered in conjunction with other immunoregulatory agents, for example, immune globulins.
D. Antibodies In another embodiment of the invention, the immunogenic polypeptides prepared as described above are used to produce antibodies, including polyclonal and monoclonal. If polyclonal antibodies are desired, a selected mammal (e.g., mouse, rabbit, sheep, goat, horse, etc.) is immunized with an immunogenic polypeptide bearing an HCV epitope(s). Serum from the immunized animal is collected and treated according to known procedures. If serum containing polyclonal antibodies to antigenic deteminant(s) comprised of the SLF--G motif contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography. Techniques for producing and processing polyclonal antisera are known in the art, see for example, Mayer and Walker (1987).
Alternatively, polyclonal antibodies may be isolated from a mammal which has been previously infected with HCV, and antibodies directed to antigenic deter<ninant(s) comprised of the SLF-G- motif isolated. Monoclonal antibodies directed against HCV epitopes can also be readily produced by one skilled in the art. The general methodology for making monoclonal antibodies by hybridomas is 0 94126306 ~ PCT/US94104853 well known. Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfe~ction with Epstein-Barr virus. See, e.g., M.
Schreier et al. (1980), HYBRIDOM:A TECHNIQUES; Hammerling et al. (1981) MONOCLONAL ANWBODIES AND T-CELL HYBRIDOMAS; Kennett et al.
(1980) MONOCLONAL AN'IZBODIES; see also, U.S. Patent Nos. 4,341,761;
4,399,121; 4,427,783; 4,444.,887; 4,466,917; 4,472,500; 4,491,632; and 4,493,890. Panels of monoclonal antibodies produced against HCV epitope(s) comprised of the SLF--G motif can be screened for various properties; i.e., for isotype, epitope affinity, etc.
Antibodies, both monoclonal and polyclonal, which are directed against HCV epitope(s) comprised of the SLF--G motif are particularly useful in diagnosis, and those which are neutralizing are useful in passive immunotherapy.
Monoclonal antibodies, in particular, may be used to raise anti-idiotype antibodies.
Anti-idiotype antibodies are immunoglobulins which carry an "internal image" of the antigen of the infectious agent against which protection is desired.
Se.e, for example, Nisonoff, A., et al., Clin. Immunol. Immunopathol. (1981) 21:397-406; and Dreesman et al., J. Infect. Disease (1985) 151:761. Techniques for raising anti-idiotype antibodies are known in the art. See, for example, GrLych, Nature (1985) 316:74; MacNamara et al., Science (1984) 226:1325; and Uytdehaag et al., J. Immunol. (1985) 134:1225. These anti-idiotype antibodies may also be useful for treatment, vaccination andlor diagnosis of HCV
infection, as well as for an elucidation of the immunogenic regions) of HCV antigens comprised of the SLF--G motif.
E. Passive Immunization In another embodiment of the invention, compositions comprised of neutralizing antibodies directed to an antigenic determinants) comprised of the SLF--G motif are used for passive immunization of individuals for prophylaxis and/or therapy of HCV infection. If the antibodies are polyclonal, it is preferable to fractionate the antibody preparations prior to administration in order to separate and concentrate active fractions, for example, inter alia, IgGs and IgMs.
Techniques for separating various fractions of antibodies are known by those of skill in the art, and require only routine methods. If monoclonal antibodies are used for passive immunization, it may be preferable to include a variety of monoclonal antibodies directed to one or more HCV antigenic determinants as well as the antibodies directed to the antigenic determinants) comprised of the SLF--G
S motif.
Methods and protocols for passive immunization are known in the art, and are discussed in several of the references cited above. Generally, the antibodies are mixed with suitable excipients. The antibodies may be given in single or multiple doses, and in effective amounts. Generally, because of differences between individuals to which the antibodies are administered, the dosage and regimen is determined by the person supervising the administration.
D. Diagnostic Assays For diagnostic application, it may be useful to employ the compositions of the present invention as antigens, thereby improving the ability to detect antibody to various HCV isolates. Typically the polypeptides can be used directly in a homogeneous or heterogeneous immunoassay format, the latter preferably comprising immobilizing the polypeptide on a solid substrate (e.g., microtiter plate wells, plastic beads, nitrocellulose, etc.). See. e.~.. PCT Publ. No.
W090111089;
EPO Publ. No. 360,088; IIUVIMUNOASSAY: A PRACTICAL GUIDE, supra.
These immunogenic compositions comprised of a polypeptide containing a region with the SLF--G motif are used to detect anti-HCV antibodies within biological samples, including for example, blood or serum samples. The immunoassay will use at least one antigen with an antigenic determinant comprised of the SLF--G
motif. It is also contemplated that antibodies directed to antigenic determinants comprised of the SLF--G motif may be used to detect antigens with the motif in biological samples. Design of the immunoassays is subject to a great deal of variation, and a variety of these are known in the art. Protocols for the immunoassay may be based, for example, upon competition, or direct reaction, or sandwich type assays. Protocols may also, for example, use solid supports, or may be by immunoprecipitation. Most assays involve the use of labeled antibody or polypeptide; the labels may be, for example, fluorescent, chemiluminescent, radioactive, or dye molecules. Assays which amplify the signals from the probe ~ 94!26306 ~ ~~ ~ .
are also known; examples of which are assays which utilize biotin and avidin, and enzyme-labeled and mediai:ed immunoassays, such as ELISA assays.
Kits suitable for immunodiagnosis and containing the appropriate labeled reagents are constructed by packaging the appropriate materials, including the 5 compositions of the inventiion in suitable containers, along with the remaining reagents and materials (for example, suitable buffers, salt solutions, etc) required for the conduct of the assay, as well as a suitable set of assay instructions.
E. Gene Theranv In another embodiment of the invention polynucleotides encoding 10 immunogenic polypeptides comprised of the SLF--G motif are used for purposes of gene therapy for individuals to prevent andlor alleviate HCV infections.
The sequence encoding the immunogenic polypeptide containing a region comprised of the SLF--G motif is operably linked to a transcriptional control region.
Transcriptional control regions are known in the art.
:5 In some embodiments of the invention, the transcriptional control regions may be hybrids, including enhancer regions, multimeric transcription factor binding sites (e.g., NF-AT' and/or NFKB), secretion signals, or positive marthers that enable the selection of cells carrying the recombinant polynucleotide.
Polynucleotide constructs prepared for introduction into a prokaryotic or 20 eukaryotic host cell for replication may comprise a replication system recognized by the host, including the :intended recombinant polynucleotide fragment encoding the desired polypeptide. Such vectors may be prepared by means of standard recombinant techniques well known in the art and discussed, for example, in Sambrook et al. (1989) or Ausubel et al. (1987).
25 The recombinant polynucleotides encoding the polypeptides of the invention may be introduced into individuals in several ways. For example, the polynucleotides may be introduced ex vivo into a host cell, for example, dendritic cells, or cells from a skin biopsy. The cells containing the recombinant polynucleotide may be usead to confer immunity to individuals. The cells are usually administered by inFusion, with each infusion in a range of at least 106 to 10'° cells/mz, preferably irn the range of at least 10' to 109 cells/m'-. The clones may be administered by a single infusion, or by multiple infusions over a range of time. However, since different individuals are expected to vary in responsiveness, the type and amount of cells infused, as well as the number of infusions and the time- range over which multiple infusions are given are determined by the attending physician or veterinarian, and can be determined by routine examination.
The polynucleotides encoding the immunogenic polypeptides comprised of the SLF--G motif may be introduced into the desired cell ex vivo by means known in the art, including, for example, transformation, electroporation, lipofection, and transduction, including the use of adeno-associated viral (AAV) vectors, and particularly using methods of retroviral gene transfer known in the art.
Various infection techniques known in the art have been developed which utilize recombinant infectious virus particles for gene delivery. Retroviral vectors provide a highly efficient method for gene transfer into eukaryotic cells.
Numerous retroviral vector constructs have been used successfully to express many foreign genes (see, e.g., Cofin, in Weiss et al. (eds. ), RNA Tumor Viruses, 2nd ed., vol. 2 (Cold Spring Harbor Laboratory, New York, 1985, pp.
17-71).
In other embodiments of the invention, the recombinant polynucleotides encoding the immunogenic polypeptides containing the SLF--G motif are introduced directly into the individual to be treated andlor immunized. In these embodiment it is preferred that the polynucleotide be in the form of an expression vector, and even more preferablyva~circular plasmid. The polynucleotides are mixed with suitable excipients, and administered to the individual by any suitable means known in the art, including, for example parenteral (including, for example, intravenous, intraperitoneal, intramuscular, and subcutaneous) ingestion, lipofection, and transdermal.
F. Examples Described below are examples provided only for illustrative purposes and not to limit the scope of the present invention. In light of the present disclosure.
numerous embodiments within the scope of the claims will be apparent to those of ordinary skill in the art. .
J 94/26306 ~ ~ . PCT/US94104853 Example 1 Identification of a Conserved Motif in E2HV
A conserved motifs) within the E2HV domain was identified by examining 90 E2HV sequences from isolates from around the world for conserved features.
The HCV sequences examined are shown in Fig. 2. The examination showed significant variability of the E2HV sequences.
E2HV sequence data from patients followed sequentially after HCV
infection is indicative that mutations appear with greater frequency between amino acids 395 to 407 and with time appear throughout the remainder of the E2HV
domain. See Fig. 3, which presents the sequence data for three patients:
Hutchinson (H) (Ogata et ;al., Proc. Natl. Acad. Sci. USA (1991) 88:3392-3396);
HC-J4 (Okamoto et al., Virolo~y (1992) 188:331-341); and NY/RS (Kato et al., Biochem. Bio~hvs. Res. C'.ommun. (1992) 181:279-285).
It is also observed from Fig. 3 that Patient RS appears to have a different pattern of fewer, randomly distributed point mutations acumulating over time.
The RS pattern of amino aicid substitutions has been observed in a subset of HCV
infected patients and in a <:hronically infected chimpanzee (See, for example, Weiner et al., Vaccines 91, supra.) An explanation for the difference in the pattern of mutations accumulated with time in patients RS and NY, for example, is that individuals such as RS fail to make antibodies to the E2HV epitope(s). In the absence of positive immure selection, the sequence mutates randomly as a quasispecies distribution of HCV variants evolves. The chronically infected chimpanzee, who had a poor immune response to HCV antigens, showed a similar pattern of E2HV mutations as patient RS.
The results of the examination of sequences is shown in Fig. 4, which shows the degree of conservation of character of amino acids 384 to 407.
Although no two EZHV sE;quences are identical, amino acids 401 to 403 and 406 to 407 are strongly conserved for the characteristics of the amino acids at those positions. Amino acid 401 is S, G, A, D, K, R, or T; amino acid 402 is L, F, I, M, or W; amino acid 403 is F or L; amino acid 406 is G or A; amino Acid 407 is A, P, or S. The relative prevalence of the amino acids in these positions in the 90 sequences examined is shown in Table 2.
WO 94!26306 - PCTIUS94/04853 Table 2. Summary of Amino Acid Substitutions in the EZHV Conserved Motif S L F G A/P/S
aa401 aa402 aa403 aa406 aa407 18G 12F 13L l A 35P
2A 6I lOS
2 T 3~ M
Example 2 Mapping of HCV1 E2HV E~itopes For epitope mapping of the E2HV region of E2/NS 1, see PCT Publ. No.
W093/00365, with the following modifications. Overlapping peptides from the sequence spanning amino acid positions 384 to 413 of HCV 1 attached to pins were prepared. The peptides were reacted with an IgG preparation from sheep that had been immunized with a conjugated 30-mer peptide from the same region.
The sheep IgG preparations containing anti-HV E2 antibodies were prepared from sheep immunized with a peptide coupled to diphtheria toxoid. The peptide spanned the HCV1 E2HV region, and had the following sequence:
acetyl-C-B-E-T-H-V-T-G-G-S-A-G-H-T-V-S-G-F-V-S-L-L-A-P-G-A-K-Q-N-V-Q-L-acid, wherein B is butyl alanine.
The results of the screening using sheep serum IgG s1634-2 and s1635-2 from sheep immunized with the conjugated 30-mer are shown in Fig. 5. The results indicate that sheep 1634-2 IgG reacts with the minumum epitope 4°°VSLLA'°~. IgG from sheep 1635-2 has a broader reactivity profile--the sera reacts with the peptides containing the minimum 4°°VSLLA4°~ epitope, and in J 94126306 PCTlUS94104853 addition, peptides containing the minimum epitopes a°'SLLAPGAa°' and 403L~GAao~. Thus, the I1;G preparation from sheep immunized with the 30-mer peptide of E2HV is reactive with linear epitope(s) between amino acids 400 to 407.
Within the conservf:d region of E2HV, the sequence of the conserved motif of HCVl is S-L-L-aa4-aa5-G-(A/P/S). The substitution of L for F in the S-L-F-aa4-aa5-G-(A/P/S) motif is conservative with respect to the amino acid characteristics.
The methods used in this example are described below.
Synthesis of overlapping_~e tn ides The synthesis of the overlapping peptides was as follows. Specially prepared and derivatized polyethylene pins arranged on a block in an 8 x 12 array (Coselco Mimotopes, Victoria, Australia) were prepared by placing the pins in a bath (20 % vlv piperidine in dimethyformamide (DMF)) for 30 minutes at room temperature. The pins were then removed,' washed in DMF for 5 min, then washed in methanol four tomes (2 minlwash). The pins were allowed to air dry for at least 10 min, then washed a final time in DMF (5 min). 1-hydroxybenzo-triazole (HOBt, 367 mg) v~ras dissolved in DMF (80 ml) for use in coupling Fmoc-protected amino acids: Frnoc-L-Ala-OPfp, Frnoc-L-Cys(Trt)-Opfp, Fmoc-L-Asp(O-tBu)-OPfp, Fmoc-t,-Glu(O-tBu)-OPfp, Fmoc-L-Phe-OPfp, Fmoc-Gly-OPfp, Fmoc-1.-His(Boc)-OPfp, Fmoc-L-Ile-OPfp, Fmoc-L-Lys(Boc)-OPfp, Fmoc-L-Leu-OPfp, Fmoc-L-Met-OPfp, Fmoc-~-Asn-OPfp, Fmoc-L-Pro-OPfp, Fmoc-L-Gin-OPfp, Fmoc-L-Arg(Mtr)-OPfp, Fmoc-L-Ser(t-Bu)-ODhbt, Fmoc-L-Thr(t-Bu)-ODhbt, Fmoc-L-Val-OPfp, and Fmoc-1.,-Tyr-Ol'fp.
The protected amino acids were placed in microtiter plate wells with HOBt, and the pin block placed ower the plate, immersing the pins in the wells. The assembly was then sealed in a plastic bag and allowed to react at 25°C
for 18 hours to couple the first amino acids to the pins. The block was then removed, and the pins washed with :DMF (2 min), MeOH (4 x 2 min), and again with DMF (2 min) to clean and. deprotect the bound amino acids. The procedure was repeated for each addition;~l amino acid coupled, until all octamers had been pre-pared. The free N-termini were then acetylated to compensate for the free amide, t'~ ~ ~~~~
as most of the epitopes an; not found at the N-terminus and thus would not have the associated positive charge. Acerylation was accomplished by filling the wells of a microtiter plate with 1DMF/acetic anhydrideltriethytamine (5:2:1 v!v!v) and allowing the pins to react in the wells for 90 min at 20°C. The pins were then 5 washed with DMF (2 min;) and MeOH (4 x 2 min), and air dried for at least 10 min.
The side chain protecting groups were removed by treating the pins with trifluoroacetic acidlphenol/dithioethane (95:2.5:2.5, v/v/v) in polypropylene bags for 4 hours at room tempe:ratun:. The pins were then washed in dichlommethane 10 (2 x 2 min), 5 ~ di-isopn~pylethylamine/dich9ommethane (2 x 5 min), dichloro-methane (5 min), and air-dried for at least 10 min. The pins were then washed in water (2 min), MeOH ( 18 hours), dried in vacuo, and stored in sealed plastic bags over silica gel.
Binding Assay 15 In order to assay binding to peptides, octamer-bearing pins prepared as described above were first treated by sonicating for 30 min in a disnrption buffer (1 ~ sodium dodecylsulfate, 0.19& 2-mercaptoethanol, 0.1 M NaH2P04) at 60°C.
The pins were then immersed several times in water (60°C), followed by boiling MeOH (2 min), and allowed to air dry.
20 The pins were then precoated for 1 hour at 25°C in miccotiter wells con-taining 200 ~.L blocking buffer (1 ~ ovalbumin, 1 °~ BSA, 0.1'6 Tween~*and 0.05 °6 NaN3 in PBS), with agitation. The pins were then immersed in microtiter wells containing two preparations of IgG obtained from sheep immunized with E2HV peptide.
25 Prelaaration of I~G containi,~ anti-HCV HV E2 antibodies The preparation of sheep IgG and of the conjugated peptide was as follows.
The sheep were immunized with 50 to 100 nmoles of the conjugated peptide in Freund's Complete Adjuvant (CFA)y 14 days later, the sheep were immunized a second time, but the conjugated peptide was in Freund's incomplete adjuvant.
30 Three to four weeks later, the sheep were bled, and IgG in the serum was precipitated with 50°.6 ammonium sulfate. The precipitate was collected and treated with a solution cotataining 1 °6 Triton X-IOCf and 0.3 ~ tri-N-butyl * Tween - trade-mark * Triton X-100 - trade-mark ~,-~~.~~55 phosphate trTIVBP). After the treatment, detergent was removed by precipitating the IgG fraction with 50 ~ ammonium sulfate, collecting and washing the precipitate twice with a sollution containing 50 ~ ammonium sulfate, followed by solubilization in and dialysis for two days against phosphate buffered saline (PBS).
The resulting IgG preparation was sterilized by passage through a filter prior to use for immunization.
Coupling of the Diphtheria. Toxaid Carrier Protein to MCS
The peptide-diphtheoria toxoid conjugates were prepared using the following protocol.
Materials:
ethylene diamine tetra-acetic acid (EDTA Na~.2Hz0) (MW 3?2) 6-maleimido-caproic acid N-hydroxysuccinimide ester (MCS) (Sigma} - 95 ~ pure sodium dihydrogen orthophosphate (NaHzPOd) nitrogen dimethylformamide (DMA
Milli Q*water O.I M phosphate buffer containing 5 mM EDTA, pH 6.66 0.1 M phosphate buffer, pla 8.0 0.1 M phosphate buffer, pl:i ?.0 sodium succinate [(CH~COONa)z.6FIz0]
cysteme hydrochloric acid (2 ~O solution) 0.1 M sodium succinatel0.1 EDTA, pH 5.6 Purified diphtheria ttoxoid (Commonwealth Serum Laboratories, Victoria, Australia) was coupled to rvICS according to the method described by Lee et al., Mol. Immunol_. (1980) X7:'149; Partis e:t al., Prot. Chem. (1983) 2:263;
Peeters et al., 1. Immunol. Methods ('1989) 120:133; (ones et al., J. Immunol. Methods (1989) 123:211. 100 ml o;F diphtheria toxoid was passed through a G25 Sephadex*
column (l7cm X 4 cm) to remove thiomersal. The toxoid was eluted with 0.1 M
phosphate buffer pH ?.0 arad the protein content of the eluate was assayed using the BCA protein determination (Pierce). The resulting solution was concentrated using an Amicon ultrafiltration unit to a final concentration of 10 mglml_ * Milli Q - trade-mark * Sephadex - trade-mark One miiliGter of the; toxoid solution was dialyzed with 0.1 M phosphate buffer, pH 8.0, and then rruixed with a solution of 1.5 mg MCS in 200 ~cl DMF.
The resulting solution was incubated at robin temperature for 1 hour in the dark with occasional mixing. In order to separate the uncoupled MCS from the MCS-'toxoid, the solution was passed through a Sephadex'~PD10 column which had been equilibrated with 0.1 M phosphate buffer, pH 6.66 and the protein fraction was collected.
. The number of maleimido groups coupled per carrier molecule was determined prior to coupling of the HCV peptides thereto. Thirty milliliters of the succinatelEDTA buffer wars sparged with nitrogen for 2 minutes. Five milligrams of cysteine was transferred into a 25 ml volumetric flask and dissolved in a final volume of 25 ml of the sparged buffer. Aliquots of the solutions shown in Table 3 were transferred in dupliczute to 25 ml screw capped bottles. Using separate pipettes, nitrogen was bubbled into each aliquot. Each bottle was then sealed and IS incubated at room temperature in the dark for ~0 minutes with occasional swirling.
T b~
Solution Sample ~ml) Standard (roll Blank ml) activated carrier 0:3 - -phosphate buffer - 0.3 0.3 cysteine solution 1.0 1:0 -succinate buffer - - I.0 * A O.I ml aliquot of each of the 3 solution was taken for an Eliman's determination.
Ellman's Test for the (quantitative Determination of Sulfhydrvl Materials Required:
Phosphate buffer, pH 8.0 Dissolve 15.6 g Na13zP04 or 12.0 g NaH=PO~ anhydrous in approximately 700 ml Milli Q*water. Adjiust the pH to 8.0 using 50 ~ NaOH. Add Milli Q~'water for a final volume of 1000 ml and then adjust the pH if necessary.
Ellman's Reagent * Sephadex - trade-mark * Milli Q - trade-mark 2 ~ 62.55 Dissolve 10.0 mg of 5,5'-diithiobis-2-nitmbenzoic acid (DTNB) in 2.5 ml of phosphate buffer, pH 8.0 0.1 ml of Ellman's reagent was added to each of the 0.1 ml aliquots of the solutions prepared above, namely the sample, standard and bland solutions.
Five milliliters of phosphate buffer, pH 8.0, was then added to each aliquot, mixed well and allowed to stamd for 15 minutes. The absorbance of each aliquot was measured in a 1 cm path length cell at 412 nm.
The number of maleimido groups present on the carrier protein was determinexi according to the; following method. A 0.01 ~.mol per ml solution of -SH produces an absorbance of 0.136 in a 1 em light path at 412 nm. The absorbance of the Standard or Sample (A) is equal to the amount of cysteine reacted with the coupled ttntleimido groups on the activated carrier protein.
Since 1 mol of available -SH reacts with 1 mol of maleimido, the concentration in ~mols of the maaeimido groups pnasent in the aliquot tested is equal to A(0.01 )!0.136 IS ~.mol/ml. The total volume: of the solution was 5.2 ml. Therefore, the total number of ~cmols present was equal to A(0.01)(5.2)10.136. The sample solution had a total volume of 1.3 ml, of which 0.3 ml consisted of the activated carrier protein. The amount of ma~leimido groups present in the sample solution was calculated as A(0.01)(5.2)(;1.3)/(0.136)(0.1)(0.3) = A(16.5"Y) ~cmoUml. The MCS-activated carrier protein was stored at -20° C.
Reduction of the HCV Peptides Prior to coupling of the HCV peptides to the MCS-activated carrier protein, the peptides were reduced to ensure that thiol groups present on the peptides were in the fully neduced -SH form.
Materials Required:
dithiothreitol (DTT) ammonium hydrogen carbonate (IVH,HC03), methanol SEP-PAK~ (C18 cartridge, Waters), 1 cartridge for each 8 mg of peptide 0.1 M ammonium hydrogen carbonate buffer Dissolve 7.9 g NH,lEiC03 in 1 L Milli Q*water Buffer A, 0.1 °~ vlv trifluoroacetic acid (TFA) in Milli ~'~ater Buffer B, 60 ~ vlv acetonitrile, 0:1 °6 vl v TFA in Mini Q'i'water * SEP-PAKs - trade-mark * Mill.i Q - trade-mark ~ 6255T
15 mg of the HCV peptide were added to 2.5 ml of 0.1 M ammonium hydrogen carbonate containing a 10 fold molar excess of DTT. The resulting solution was mixed until the peptide had dissolved and was then allowed to stand for 1 hour at room temperature. A pair of SEP-PAKs were connected in series S and activated by passing approximately 20 ml of methanol and then 20 ml of Buffer A through the pair of SEP-PAK~: ' The peptidelDTT sample was slowly passed through a pair of S:EP-PAKS* The DTT was eluted with approximately 20 ml of Buffer A. The reducxd peptide was eluted with 7 ml of Buffer B into a pre-weighed bottle and then fn~xze-dried overnight. The bottles were then weighed to IO determined the amount of recovered peptide. The reduced peptide was then immediately coupled to thf; MCS-activated carrier protein.
CouDing HCV Peptides to MCS-Activated Carrier Protein Approximately 100 ml of 0.1 M phosphate buffer with 5 mM FrDTA, pH
6.66 was degassed under vacuum and then sparged with nitrogen for 10 minutes.
15 Twenty milliliters of a IO mg/ml solution of the MCS-activated carrier protein was carefully sparged with nitrogen to prevent excessive frothing. 5 mg of the reduced peptide were dissolved in approximately 0.2 ml of the degassed sparged phosphatelEDTA buffer, p~H 6.66 and then mixed with the MCS-activated carrier protein solution. The resulting mixture was transferred into a screw capped bottle 20 which was then filled with nitrogen and sealed. The solution was further degassed by holding the bottle in a ',Branson 2000~ sonication bath for 2 minutes. The bottle was covered with aluminum foil and incubated overnight at room temperature with slow mixing on a shaker table.
The resultant conjugate was soluble and the uncoupled peptide was 25 removed by passing the mixture over a Sephadex*PD 10 column which had been equilibrated with the phosl~hate/EDTA buffer, pH 6.66. The protein fraction was .
collected. The amount of peptide conjugated to the carrier protein was determined by amino acid analysis.
An amino acid analysis of 150 gel aliquots of both the conjugate and the 30 carrier protein was performed. The average ratio of the level of amino acids contributed solely by the carrier protein was determined to calculate the amount of conjugated peptide produced. Levels of serine, threonine, tryptophan.
methionine.
* SEP-PAKs - trade-mark * Sephadex - trade-mark 'O 94/26306 ~ PCT/US94104853 tyrosine and cysteine were :not determined as these amino acids are modified under the standard hydrolysis conditions.
Example 3 5 Binding of Anti-thyroxin Monoclonal Antibodies to HCV E2HV Domain Peptides Monoclonal antibodiies that bind to thyroxine (T4) were prepared by Dr.
Mario Geysen, Chiron Mimitopes Ltd, Australia. The binding of these antibodies to overlapping peptides that: span the F,2HV region was assessed. Peptides on pins 10 were prepared essentially a;s described above in Example 2, except that the HCV
E2HV sequence spanned from amino acid 383 to 413 of HCV 1. The binding of the anti-T4 monoclonal antibodies to the HCV E2HV mimitopes was performed in duplicate. The binding results are shown in the bar graph in Fig. 6, where the solid and shaded bars represent binding of each of the duplicate samples. As seen 15 in the figures, the anti-T4 antibodies were immunologically reactive with epitope(s) encompassed within the HCV 1 sequence that spanned from aa395 to aa407.
Example 4 20 Binding of Serum Proteins to HCV
The binding of thref: serum proteins, human prealbumin, human serum albumin, and thyroid binding globulin (TBG) to overlapping peptides spanning E2HV was performed. Octamer-bearing pins were prepared as described in Example 1. The binding of the designated serum proteins to the octamers was 25 determined by an ELISA a;~say, using antibodies directed to the specific proteins.
Controls were run in the absence of the serum proteins but in the presence of the respective antibodies. The results, shown as difference plots, are shown in Fig. 7.
Based upon the results, it appears that transthyretin binds to at least one minimum epitope in the hypervariabl~~ region. Yn addition, the results are suggestive that 30 TBG binds to two minimurn epitopes, one of which encompasses the SLF--G
motif.
Preliminary evidence suggests that the hypervariable domains) of E2/NS 1 may be responsible for escape mutants, leading to chronic HCV infections.
However, a conserved region within the hypervariable region is suggestive that the conserved region has an important function and plays an essential role in virus binding andlor entry into and/or replication in the host cell. In virus binding it is contemplated that the binding may be to the cell and/or to another molecule which facilitates virus binding and/or entry and/or replication. The examples presented infra. are suggestive that virus binding to transthyretin and/or to thyroid binding globulin (TBG) are involved in the infective process. Thus, increasing an immune response to antigenic determinants containing the conserved SLF--G sequence may lead not only to protection against and/or alleviation of HCV infection, but also to a reduction in chronicity of HCV infection. In addition, the conserved region is also suggestive that the vaccines comprised of the immununoreactive polypeptides having a region with the SLF--G motif may be cross-reactive.
In preferred applications for vaccines, the polypeptide compositions described herein are combined with other HCV subunit antigens, for example, those described in PCT Publ. No. W092/08734. In cases where the composition is to be used for treatment of HCV, it is desirable that the composition be immunogenic. In instances wherein the synthesized polypeptide is correctly configured so as to provide the correct epitope, but is too small to be immunogenic, the polypeptide may be linked to a suitable Garner. A number of techniques for obtaining such linkage are known in the art, including the formation of disulfide linkages using N-succinimidyl-3-(2-pyridyl-thio)propionate (SPDP) and succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) (if the peptide lacks a sulfhydryl group, this can be provided by addition of a cysteine !O 94126306 residue. ) These reagents create a disulfide linkage between themselves and peptide cysteine resides on one protein and an amide linkage through the e-amino on a lysine, or other free amino group in other amino acids. A variety of such disulfide/amide-forming agents are known. See, for example, Immun. Rev.
( 1982) 62:185. Other bifunctional coupling agents for a thioether rather than a disulfide linkage. Many of these thio-ether-forming agents are commercially available and include reactive esters of 6-maleimidocaproic acid, 2-bromoacetic acid, 2-iodoacetic acid, 4-((~T-maleimido-methyl)cyclohexane-1-carboxylic acid, and the like. The carboxyl groups can be activated by combining them with succinimide or 1-hydroxyl-2-nitro-4-sulfonic acid, sodium salt. Additional methods of coupling antigens employ the rotavirus/°'binding peptide"
system described in EPO Publ. No. 259,149. The foregoing list is not meant to be exhaustive, and modifications of the named compounds can clearly be used.
Any carrier may be used which does not itself induce the production of antibodies harmful to the host. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins; polysaccharides such as latex functionalized sepharose, a;garose, cellulose, cellulose beads and the like;
polymeric amino acids, such as polyglutamic acid, polylysine, and the like;
amino acid copolymers; and inactive virus particles (see infra.). Especially useful protein substrates are serum albumins, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, crvalbumin, tetanus toxoid, and other proteins well known to those of skill in t:he art.
The immunogenicity of the antigens comprised of the SLF--G motif may also be enhanced by prepaung them in eukaryotic systems fused with or assembled with particle-forming proteins such as, for example, that associated with hepatitis B surface antigen. See, e.;~., U.S. Patent No. 4,722,840. These constructs may also be expressed in mammalian cells such as CHO cells using an SV40-dihydrofolate reductase vector (Michelle et al. ( 1984), INTERNATIONAL
SYMPOSIUM ON VIRAL HEPATITIS).
In addition, portions of the particle-forming protein coding sequence may be replaced with codons encoding the SLF--G epitope from an HCV hypervariable domain. In this replacement, regions which are not required to mediate the 20 2 ~ 6~55~
aggregation of the units to form immunogenic particles in yeast yr mammals can be deleted, thus eliminating additional HBV antigenic sites from competition with the HCV epitope(s).
These vaccines ma~,y either be prophylactic (to prevent infection) or therapeutic (to treat disea:~e after infection).
Such vaccines comprise antigen or antigens, usually in combination with "pharmaceutically acceptable carriers," which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles. Such carriers are well known to those of ordinary skill iin the aut. Additionally, these carriers may function as immunostimulating agents ("adjuvants"). Furthermore, the antigen may be conjugated to a bacterial toxoid, such as a toxoid from diphtheria, tetanus, cholera, -~I , etc. pathogens.
Preferred adjuvant;s to enhance effectiveness of the composition include, but are not limited to: (1) aauminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aaunninum sulfate, etc; (2) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides (see below) or bacterial cell wall components), such as for example (a) MF59 (PCT Publ. No. WO 90114837), containing 5 % Squalene* 0.5 % Tween*80, and 0.5 ~o Span 85'"(optior.~ally containing various amounts of MTP-PE (see below), although not required) formulated into submicmn particles ,using a microfluidizer such as Model llOY microfluidizer (Microfluidics, Newton, MA), (b) SAF, containing 10'~ Squalane;k0.4 ~ Tweeri 80, 5 % pluronic-blocked polymer Ll 21, and thr-MDP (see below) either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) R.ibrT'"
adjuvant system (RAS), (Ribi Imm~unochem, Hamilton, MT) containing 2 % Squalen~, 0.2 Tween*$0, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate ('TDM), and cell wall skeleton (CWS), prederably MPL + CWS (DetoxT"'); (3) saponin adjuvants, * Tween - trade-mark * Span 85 - trade-mark *Squalane - trade-mark * Squalene - trade-mark O 94/26306 ~ ~ PCTlUS94104853 such as StimulonTM (Cambridge Bioscience, Worcester, MA) may be used or particles generated therefrom such as ISCOMs (immunostimulating complexes);
(4) Complete Freunds Adjuvant (CFA) and Incomplete Freunds Adjuvant (IFA);
(5) cytokines, such as interleukins (IL-l, IL-2, etc.), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc; and (6) other substances that act as immunostimulating agents to enhance the effectiveness of the composition. Alum and N1F59 are preferred.
As mentioned above, muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-( 1 '-2'-dipalinitoyl-sn-glycero-3-hydroxyphosphoryioxy)-ethylamine (MTP-PE), etc.
The immunogenic compositions (e.g., the antigen, pharmaceutically acceptable carrier, and adjuvant) typically will contain diluents, such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.
Typically, the imm unogenic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation also may be emulsified or encapsulated in liposomes for enhanced adjuvant effect, as discussf:d above under pharmaceutically acceptable carriers.
Immunogenic compositions used as vaccines comprise an immunologically effective amount of the antigenic polypeptides, as well as any other of the above-mentioned components, as needed. "Immunologically effective amount" means that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment. This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated (e. g. , nonhuman primate, primate, etc. ), the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the WO 94126306 PCTlUS94104853 medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
The immunogenic compositions are conventionally administered parenterally, e.g., by injection, either subcutaneously or intramuscularly.
Additional formulations suitable for other modes of administration include oral and pulinonary formulations, suppositories, and transdermal applications. Dosage treatment may be a single dose schedule or a multiple dose schedule. The vaccine may be administered in conjunction with other immunoregulatory agents.
In addition to the above, it is also possible to prepare live vaccines of attenuated microorganisms which express recombinant polypeptides comprised of a region with the SLF--G motif. Suitable attenuated microorganisms are known in the art and include, for example, viruses (e.g., vaccinia virus) as well as bacteria.
In addition, the vaccine containing the polypeptide with an antigenic determinant comprised of the conserved motif SLF--G may be administered in conjunction with other immunoregulatory agents, for example, immune globulins.
D. Antibodies In another embodiment of the invention, the immunogenic polypeptides prepared as described above are used to produce antibodies, including polyclonal and monoclonal. If polyclonal antibodies are desired, a selected mammal (e.g., mouse, rabbit, sheep, goat, horse, etc.) is immunized with an immunogenic polypeptide bearing an HCV epitope(s). Serum from the immunized animal is collected and treated according to known procedures. If serum containing polyclonal antibodies to antigenic deteminant(s) comprised of the SLF--G motif contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography. Techniques for producing and processing polyclonal antisera are known in the art, see for example, Mayer and Walker (1987).
Alternatively, polyclonal antibodies may be isolated from a mammal which has been previously infected with HCV, and antibodies directed to antigenic deter<ninant(s) comprised of the SLF-G- motif isolated. Monoclonal antibodies directed against HCV epitopes can also be readily produced by one skilled in the art. The general methodology for making monoclonal antibodies by hybridomas is 0 94126306 ~ PCT/US94104853 well known. Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfe~ction with Epstein-Barr virus. See, e.g., M.
Schreier et al. (1980), HYBRIDOM:A TECHNIQUES; Hammerling et al. (1981) MONOCLONAL ANWBODIES AND T-CELL HYBRIDOMAS; Kennett et al.
(1980) MONOCLONAL AN'IZBODIES; see also, U.S. Patent Nos. 4,341,761;
4,399,121; 4,427,783; 4,444.,887; 4,466,917; 4,472,500; 4,491,632; and 4,493,890. Panels of monoclonal antibodies produced against HCV epitope(s) comprised of the SLF--G motif can be screened for various properties; i.e., for isotype, epitope affinity, etc.
Antibodies, both monoclonal and polyclonal, which are directed against HCV epitope(s) comprised of the SLF--G motif are particularly useful in diagnosis, and those which are neutralizing are useful in passive immunotherapy.
Monoclonal antibodies, in particular, may be used to raise anti-idiotype antibodies.
Anti-idiotype antibodies are immunoglobulins which carry an "internal image" of the antigen of the infectious agent against which protection is desired.
Se.e, for example, Nisonoff, A., et al., Clin. Immunol. Immunopathol. (1981) 21:397-406; and Dreesman et al., J. Infect. Disease (1985) 151:761. Techniques for raising anti-idiotype antibodies are known in the art. See, for example, GrLych, Nature (1985) 316:74; MacNamara et al., Science (1984) 226:1325; and Uytdehaag et al., J. Immunol. (1985) 134:1225. These anti-idiotype antibodies may also be useful for treatment, vaccination andlor diagnosis of HCV
infection, as well as for an elucidation of the immunogenic regions) of HCV antigens comprised of the SLF--G motif.
E. Passive Immunization In another embodiment of the invention, compositions comprised of neutralizing antibodies directed to an antigenic determinants) comprised of the SLF--G motif are used for passive immunization of individuals for prophylaxis and/or therapy of HCV infection. If the antibodies are polyclonal, it is preferable to fractionate the antibody preparations prior to administration in order to separate and concentrate active fractions, for example, inter alia, IgGs and IgMs.
Techniques for separating various fractions of antibodies are known by those of skill in the art, and require only routine methods. If monoclonal antibodies are used for passive immunization, it may be preferable to include a variety of monoclonal antibodies directed to one or more HCV antigenic determinants as well as the antibodies directed to the antigenic determinants) comprised of the SLF--G
S motif.
Methods and protocols for passive immunization are known in the art, and are discussed in several of the references cited above. Generally, the antibodies are mixed with suitable excipients. The antibodies may be given in single or multiple doses, and in effective amounts. Generally, because of differences between individuals to which the antibodies are administered, the dosage and regimen is determined by the person supervising the administration.
D. Diagnostic Assays For diagnostic application, it may be useful to employ the compositions of the present invention as antigens, thereby improving the ability to detect antibody to various HCV isolates. Typically the polypeptides can be used directly in a homogeneous or heterogeneous immunoassay format, the latter preferably comprising immobilizing the polypeptide on a solid substrate (e.g., microtiter plate wells, plastic beads, nitrocellulose, etc.). See. e.~.. PCT Publ. No.
W090111089;
EPO Publ. No. 360,088; IIUVIMUNOASSAY: A PRACTICAL GUIDE, supra.
These immunogenic compositions comprised of a polypeptide containing a region with the SLF--G motif are used to detect anti-HCV antibodies within biological samples, including for example, blood or serum samples. The immunoassay will use at least one antigen with an antigenic determinant comprised of the SLF--G
motif. It is also contemplated that antibodies directed to antigenic determinants comprised of the SLF--G motif may be used to detect antigens with the motif in biological samples. Design of the immunoassays is subject to a great deal of variation, and a variety of these are known in the art. Protocols for the immunoassay may be based, for example, upon competition, or direct reaction, or sandwich type assays. Protocols may also, for example, use solid supports, or may be by immunoprecipitation. Most assays involve the use of labeled antibody or polypeptide; the labels may be, for example, fluorescent, chemiluminescent, radioactive, or dye molecules. Assays which amplify the signals from the probe ~ 94!26306 ~ ~~ ~ .
are also known; examples of which are assays which utilize biotin and avidin, and enzyme-labeled and mediai:ed immunoassays, such as ELISA assays.
Kits suitable for immunodiagnosis and containing the appropriate labeled reagents are constructed by packaging the appropriate materials, including the 5 compositions of the inventiion in suitable containers, along with the remaining reagents and materials (for example, suitable buffers, salt solutions, etc) required for the conduct of the assay, as well as a suitable set of assay instructions.
E. Gene Theranv In another embodiment of the invention polynucleotides encoding 10 immunogenic polypeptides comprised of the SLF--G motif are used for purposes of gene therapy for individuals to prevent andlor alleviate HCV infections.
The sequence encoding the immunogenic polypeptide containing a region comprised of the SLF--G motif is operably linked to a transcriptional control region.
Transcriptional control regions are known in the art.
:5 In some embodiments of the invention, the transcriptional control regions may be hybrids, including enhancer regions, multimeric transcription factor binding sites (e.g., NF-AT' and/or NFKB), secretion signals, or positive marthers that enable the selection of cells carrying the recombinant polynucleotide.
Polynucleotide constructs prepared for introduction into a prokaryotic or 20 eukaryotic host cell for replication may comprise a replication system recognized by the host, including the :intended recombinant polynucleotide fragment encoding the desired polypeptide. Such vectors may be prepared by means of standard recombinant techniques well known in the art and discussed, for example, in Sambrook et al. (1989) or Ausubel et al. (1987).
25 The recombinant polynucleotides encoding the polypeptides of the invention may be introduced into individuals in several ways. For example, the polynucleotides may be introduced ex vivo into a host cell, for example, dendritic cells, or cells from a skin biopsy. The cells containing the recombinant polynucleotide may be usead to confer immunity to individuals. The cells are usually administered by inFusion, with each infusion in a range of at least 106 to 10'° cells/mz, preferably irn the range of at least 10' to 109 cells/m'-. The clones may be administered by a single infusion, or by multiple infusions over a range of time. However, since different individuals are expected to vary in responsiveness, the type and amount of cells infused, as well as the number of infusions and the time- range over which multiple infusions are given are determined by the attending physician or veterinarian, and can be determined by routine examination.
The polynucleotides encoding the immunogenic polypeptides comprised of the SLF--G motif may be introduced into the desired cell ex vivo by means known in the art, including, for example, transformation, electroporation, lipofection, and transduction, including the use of adeno-associated viral (AAV) vectors, and particularly using methods of retroviral gene transfer known in the art.
Various infection techniques known in the art have been developed which utilize recombinant infectious virus particles for gene delivery. Retroviral vectors provide a highly efficient method for gene transfer into eukaryotic cells.
Numerous retroviral vector constructs have been used successfully to express many foreign genes (see, e.g., Cofin, in Weiss et al. (eds. ), RNA Tumor Viruses, 2nd ed., vol. 2 (Cold Spring Harbor Laboratory, New York, 1985, pp.
17-71).
In other embodiments of the invention, the recombinant polynucleotides encoding the immunogenic polypeptides containing the SLF--G motif are introduced directly into the individual to be treated andlor immunized. In these embodiment it is preferred that the polynucleotide be in the form of an expression vector, and even more preferablyva~circular plasmid. The polynucleotides are mixed with suitable excipients, and administered to the individual by any suitable means known in the art, including, for example parenteral (including, for example, intravenous, intraperitoneal, intramuscular, and subcutaneous) ingestion, lipofection, and transdermal.
F. Examples Described below are examples provided only for illustrative purposes and not to limit the scope of the present invention. In light of the present disclosure.
numerous embodiments within the scope of the claims will be apparent to those of ordinary skill in the art. .
J 94/26306 ~ ~ . PCT/US94104853 Example 1 Identification of a Conserved Motif in E2HV
A conserved motifs) within the E2HV domain was identified by examining 90 E2HV sequences from isolates from around the world for conserved features.
The HCV sequences examined are shown in Fig. 2. The examination showed significant variability of the E2HV sequences.
E2HV sequence data from patients followed sequentially after HCV
infection is indicative that mutations appear with greater frequency between amino acids 395 to 407 and with time appear throughout the remainder of the E2HV
domain. See Fig. 3, which presents the sequence data for three patients:
Hutchinson (H) (Ogata et ;al., Proc. Natl. Acad. Sci. USA (1991) 88:3392-3396);
HC-J4 (Okamoto et al., Virolo~y (1992) 188:331-341); and NY/RS (Kato et al., Biochem. Bio~hvs. Res. C'.ommun. (1992) 181:279-285).
It is also observed from Fig. 3 that Patient RS appears to have a different pattern of fewer, randomly distributed point mutations acumulating over time.
The RS pattern of amino aicid substitutions has been observed in a subset of HCV
infected patients and in a <:hronically infected chimpanzee (See, for example, Weiner et al., Vaccines 91, supra.) An explanation for the difference in the pattern of mutations accumulated with time in patients RS and NY, for example, is that individuals such as RS fail to make antibodies to the E2HV epitope(s). In the absence of positive immure selection, the sequence mutates randomly as a quasispecies distribution of HCV variants evolves. The chronically infected chimpanzee, who had a poor immune response to HCV antigens, showed a similar pattern of E2HV mutations as patient RS.
The results of the examination of sequences is shown in Fig. 4, which shows the degree of conservation of character of amino acids 384 to 407.
Although no two EZHV sE;quences are identical, amino acids 401 to 403 and 406 to 407 are strongly conserved for the characteristics of the amino acids at those positions. Amino acid 401 is S, G, A, D, K, R, or T; amino acid 402 is L, F, I, M, or W; amino acid 403 is F or L; amino acid 406 is G or A; amino Acid 407 is A, P, or S. The relative prevalence of the amino acids in these positions in the 90 sequences examined is shown in Table 2.
WO 94!26306 - PCTIUS94/04853 Table 2. Summary of Amino Acid Substitutions in the EZHV Conserved Motif S L F G A/P/S
aa401 aa402 aa403 aa406 aa407 18G 12F 13L l A 35P
2A 6I lOS
2 T 3~ M
Example 2 Mapping of HCV1 E2HV E~itopes For epitope mapping of the E2HV region of E2/NS 1, see PCT Publ. No.
W093/00365, with the following modifications. Overlapping peptides from the sequence spanning amino acid positions 384 to 413 of HCV 1 attached to pins were prepared. The peptides were reacted with an IgG preparation from sheep that had been immunized with a conjugated 30-mer peptide from the same region.
The sheep IgG preparations containing anti-HV E2 antibodies were prepared from sheep immunized with a peptide coupled to diphtheria toxoid. The peptide spanned the HCV1 E2HV region, and had the following sequence:
acetyl-C-B-E-T-H-V-T-G-G-S-A-G-H-T-V-S-G-F-V-S-L-L-A-P-G-A-K-Q-N-V-Q-L-acid, wherein B is butyl alanine.
The results of the screening using sheep serum IgG s1634-2 and s1635-2 from sheep immunized with the conjugated 30-mer are shown in Fig. 5. The results indicate that sheep 1634-2 IgG reacts with the minumum epitope 4°°VSLLA'°~. IgG from sheep 1635-2 has a broader reactivity profile--the sera reacts with the peptides containing the minimum 4°°VSLLA4°~ epitope, and in J 94126306 PCTlUS94104853 addition, peptides containing the minimum epitopes a°'SLLAPGAa°' and 403L~GAao~. Thus, the I1;G preparation from sheep immunized with the 30-mer peptide of E2HV is reactive with linear epitope(s) between amino acids 400 to 407.
Within the conservf:d region of E2HV, the sequence of the conserved motif of HCVl is S-L-L-aa4-aa5-G-(A/P/S). The substitution of L for F in the S-L-F-aa4-aa5-G-(A/P/S) motif is conservative with respect to the amino acid characteristics.
The methods used in this example are described below.
Synthesis of overlapping_~e tn ides The synthesis of the overlapping peptides was as follows. Specially prepared and derivatized polyethylene pins arranged on a block in an 8 x 12 array (Coselco Mimotopes, Victoria, Australia) were prepared by placing the pins in a bath (20 % vlv piperidine in dimethyformamide (DMF)) for 30 minutes at room temperature. The pins were then removed,' washed in DMF for 5 min, then washed in methanol four tomes (2 minlwash). The pins were allowed to air dry for at least 10 min, then washed a final time in DMF (5 min). 1-hydroxybenzo-triazole (HOBt, 367 mg) v~ras dissolved in DMF (80 ml) for use in coupling Fmoc-protected amino acids: Frnoc-L-Ala-OPfp, Frnoc-L-Cys(Trt)-Opfp, Fmoc-L-Asp(O-tBu)-OPfp, Fmoc-t,-Glu(O-tBu)-OPfp, Fmoc-L-Phe-OPfp, Fmoc-Gly-OPfp, Fmoc-1.-His(Boc)-OPfp, Fmoc-L-Ile-OPfp, Fmoc-L-Lys(Boc)-OPfp, Fmoc-L-Leu-OPfp, Fmoc-L-Met-OPfp, Fmoc-~-Asn-OPfp, Fmoc-L-Pro-OPfp, Fmoc-L-Gin-OPfp, Fmoc-L-Arg(Mtr)-OPfp, Fmoc-L-Ser(t-Bu)-ODhbt, Fmoc-L-Thr(t-Bu)-ODhbt, Fmoc-L-Val-OPfp, and Fmoc-1.,-Tyr-Ol'fp.
The protected amino acids were placed in microtiter plate wells with HOBt, and the pin block placed ower the plate, immersing the pins in the wells. The assembly was then sealed in a plastic bag and allowed to react at 25°C
for 18 hours to couple the first amino acids to the pins. The block was then removed, and the pins washed with :DMF (2 min), MeOH (4 x 2 min), and again with DMF (2 min) to clean and. deprotect the bound amino acids. The procedure was repeated for each addition;~l amino acid coupled, until all octamers had been pre-pared. The free N-termini were then acetylated to compensate for the free amide, t'~ ~ ~~~~
as most of the epitopes an; not found at the N-terminus and thus would not have the associated positive charge. Acerylation was accomplished by filling the wells of a microtiter plate with 1DMF/acetic anhydrideltriethytamine (5:2:1 v!v!v) and allowing the pins to react in the wells for 90 min at 20°C. The pins were then 5 washed with DMF (2 min;) and MeOH (4 x 2 min), and air dried for at least 10 min.
The side chain protecting groups were removed by treating the pins with trifluoroacetic acidlphenol/dithioethane (95:2.5:2.5, v/v/v) in polypropylene bags for 4 hours at room tempe:ratun:. The pins were then washed in dichlommethane 10 (2 x 2 min), 5 ~ di-isopn~pylethylamine/dich9ommethane (2 x 5 min), dichloro-methane (5 min), and air-dried for at least 10 min. The pins were then washed in water (2 min), MeOH ( 18 hours), dried in vacuo, and stored in sealed plastic bags over silica gel.
Binding Assay 15 In order to assay binding to peptides, octamer-bearing pins prepared as described above were first treated by sonicating for 30 min in a disnrption buffer (1 ~ sodium dodecylsulfate, 0.19& 2-mercaptoethanol, 0.1 M NaH2P04) at 60°C.
The pins were then immersed several times in water (60°C), followed by boiling MeOH (2 min), and allowed to air dry.
20 The pins were then precoated for 1 hour at 25°C in miccotiter wells con-taining 200 ~.L blocking buffer (1 ~ ovalbumin, 1 °~ BSA, 0.1'6 Tween~*and 0.05 °6 NaN3 in PBS), with agitation. The pins were then immersed in microtiter wells containing two preparations of IgG obtained from sheep immunized with E2HV peptide.
25 Prelaaration of I~G containi,~ anti-HCV HV E2 antibodies The preparation of sheep IgG and of the conjugated peptide was as follows.
The sheep were immunized with 50 to 100 nmoles of the conjugated peptide in Freund's Complete Adjuvant (CFA)y 14 days later, the sheep were immunized a second time, but the conjugated peptide was in Freund's incomplete adjuvant.
30 Three to four weeks later, the sheep were bled, and IgG in the serum was precipitated with 50°.6 ammonium sulfate. The precipitate was collected and treated with a solution cotataining 1 °6 Triton X-IOCf and 0.3 ~ tri-N-butyl * Tween - trade-mark * Triton X-100 - trade-mark ~,-~~.~~55 phosphate trTIVBP). After the treatment, detergent was removed by precipitating the IgG fraction with 50 ~ ammonium sulfate, collecting and washing the precipitate twice with a sollution containing 50 ~ ammonium sulfate, followed by solubilization in and dialysis for two days against phosphate buffered saline (PBS).
The resulting IgG preparation was sterilized by passage through a filter prior to use for immunization.
Coupling of the Diphtheria. Toxaid Carrier Protein to MCS
The peptide-diphtheoria toxoid conjugates were prepared using the following protocol.
Materials:
ethylene diamine tetra-acetic acid (EDTA Na~.2Hz0) (MW 3?2) 6-maleimido-caproic acid N-hydroxysuccinimide ester (MCS) (Sigma} - 95 ~ pure sodium dihydrogen orthophosphate (NaHzPOd) nitrogen dimethylformamide (DMA
Milli Q*water O.I M phosphate buffer containing 5 mM EDTA, pH 6.66 0.1 M phosphate buffer, pla 8.0 0.1 M phosphate buffer, pl:i ?.0 sodium succinate [(CH~COONa)z.6FIz0]
cysteme hydrochloric acid (2 ~O solution) 0.1 M sodium succinatel0.1 EDTA, pH 5.6 Purified diphtheria ttoxoid (Commonwealth Serum Laboratories, Victoria, Australia) was coupled to rvICS according to the method described by Lee et al., Mol. Immunol_. (1980) X7:'149; Partis e:t al., Prot. Chem. (1983) 2:263;
Peeters et al., 1. Immunol. Methods ('1989) 120:133; (ones et al., J. Immunol. Methods (1989) 123:211. 100 ml o;F diphtheria toxoid was passed through a G25 Sephadex*
column (l7cm X 4 cm) to remove thiomersal. The toxoid was eluted with 0.1 M
phosphate buffer pH ?.0 arad the protein content of the eluate was assayed using the BCA protein determination (Pierce). The resulting solution was concentrated using an Amicon ultrafiltration unit to a final concentration of 10 mglml_ * Milli Q - trade-mark * Sephadex - trade-mark One miiliGter of the; toxoid solution was dialyzed with 0.1 M phosphate buffer, pH 8.0, and then rruixed with a solution of 1.5 mg MCS in 200 ~cl DMF.
The resulting solution was incubated at robin temperature for 1 hour in the dark with occasional mixing. In order to separate the uncoupled MCS from the MCS-'toxoid, the solution was passed through a Sephadex'~PD10 column which had been equilibrated with 0.1 M phosphate buffer, pH 6.66 and the protein fraction was collected.
. The number of maleimido groups coupled per carrier molecule was determined prior to coupling of the HCV peptides thereto. Thirty milliliters of the succinatelEDTA buffer wars sparged with nitrogen for 2 minutes. Five milligrams of cysteine was transferred into a 25 ml volumetric flask and dissolved in a final volume of 25 ml of the sparged buffer. Aliquots of the solutions shown in Table 3 were transferred in dupliczute to 25 ml screw capped bottles. Using separate pipettes, nitrogen was bubbled into each aliquot. Each bottle was then sealed and IS incubated at room temperature in the dark for ~0 minutes with occasional swirling.
T b~
Solution Sample ~ml) Standard (roll Blank ml) activated carrier 0:3 - -phosphate buffer - 0.3 0.3 cysteine solution 1.0 1:0 -succinate buffer - - I.0 * A O.I ml aliquot of each of the 3 solution was taken for an Eliman's determination.
Ellman's Test for the (quantitative Determination of Sulfhydrvl Materials Required:
Phosphate buffer, pH 8.0 Dissolve 15.6 g Na13zP04 or 12.0 g NaH=PO~ anhydrous in approximately 700 ml Milli Q*water. Adjiust the pH to 8.0 using 50 ~ NaOH. Add Milli Q~'water for a final volume of 1000 ml and then adjust the pH if necessary.
Ellman's Reagent * Sephadex - trade-mark * Milli Q - trade-mark 2 ~ 62.55 Dissolve 10.0 mg of 5,5'-diithiobis-2-nitmbenzoic acid (DTNB) in 2.5 ml of phosphate buffer, pH 8.0 0.1 ml of Ellman's reagent was added to each of the 0.1 ml aliquots of the solutions prepared above, namely the sample, standard and bland solutions.
Five milliliters of phosphate buffer, pH 8.0, was then added to each aliquot, mixed well and allowed to stamd for 15 minutes. The absorbance of each aliquot was measured in a 1 cm path length cell at 412 nm.
The number of maleimido groups present on the carrier protein was determinexi according to the; following method. A 0.01 ~.mol per ml solution of -SH produces an absorbance of 0.136 in a 1 em light path at 412 nm. The absorbance of the Standard or Sample (A) is equal to the amount of cysteine reacted with the coupled ttntleimido groups on the activated carrier protein.
Since 1 mol of available -SH reacts with 1 mol of maleimido, the concentration in ~mols of the maaeimido groups pnasent in the aliquot tested is equal to A(0.01 )!0.136 IS ~.mol/ml. The total volume: of the solution was 5.2 ml. Therefore, the total number of ~cmols present was equal to A(0.01)(5.2)10.136. The sample solution had a total volume of 1.3 ml, of which 0.3 ml consisted of the activated carrier protein. The amount of ma~leimido groups present in the sample solution was calculated as A(0.01)(5.2)(;1.3)/(0.136)(0.1)(0.3) = A(16.5"Y) ~cmoUml. The MCS-activated carrier protein was stored at -20° C.
Reduction of the HCV Peptides Prior to coupling of the HCV peptides to the MCS-activated carrier protein, the peptides were reduced to ensure that thiol groups present on the peptides were in the fully neduced -SH form.
Materials Required:
dithiothreitol (DTT) ammonium hydrogen carbonate (IVH,HC03), methanol SEP-PAK~ (C18 cartridge, Waters), 1 cartridge for each 8 mg of peptide 0.1 M ammonium hydrogen carbonate buffer Dissolve 7.9 g NH,lEiC03 in 1 L Milli Q*water Buffer A, 0.1 °~ vlv trifluoroacetic acid (TFA) in Milli ~'~ater Buffer B, 60 ~ vlv acetonitrile, 0:1 °6 vl v TFA in Mini Q'i'water * SEP-PAKs - trade-mark * Mill.i Q - trade-mark ~ 6255T
15 mg of the HCV peptide were added to 2.5 ml of 0.1 M ammonium hydrogen carbonate containing a 10 fold molar excess of DTT. The resulting solution was mixed until the peptide had dissolved and was then allowed to stand for 1 hour at room temperature. A pair of SEP-PAKs were connected in series S and activated by passing approximately 20 ml of methanol and then 20 ml of Buffer A through the pair of SEP-PAK~: ' The peptidelDTT sample was slowly passed through a pair of S:EP-PAKS* The DTT was eluted with approximately 20 ml of Buffer A. The reducxd peptide was eluted with 7 ml of Buffer B into a pre-weighed bottle and then fn~xze-dried overnight. The bottles were then weighed to IO determined the amount of recovered peptide. The reduced peptide was then immediately coupled to thf; MCS-activated carrier protein.
CouDing HCV Peptides to MCS-Activated Carrier Protein Approximately 100 ml of 0.1 M phosphate buffer with 5 mM FrDTA, pH
6.66 was degassed under vacuum and then sparged with nitrogen for 10 minutes.
15 Twenty milliliters of a IO mg/ml solution of the MCS-activated carrier protein was carefully sparged with nitrogen to prevent excessive frothing. 5 mg of the reduced peptide were dissolved in approximately 0.2 ml of the degassed sparged phosphatelEDTA buffer, p~H 6.66 and then mixed with the MCS-activated carrier protein solution. The resulting mixture was transferred into a screw capped bottle 20 which was then filled with nitrogen and sealed. The solution was further degassed by holding the bottle in a ',Branson 2000~ sonication bath for 2 minutes. The bottle was covered with aluminum foil and incubated overnight at room temperature with slow mixing on a shaker table.
The resultant conjugate was soluble and the uncoupled peptide was 25 removed by passing the mixture over a Sephadex*PD 10 column which had been equilibrated with the phosl~hate/EDTA buffer, pH 6.66. The protein fraction was .
collected. The amount of peptide conjugated to the carrier protein was determined by amino acid analysis.
An amino acid analysis of 150 gel aliquots of both the conjugate and the 30 carrier protein was performed. The average ratio of the level of amino acids contributed solely by the carrier protein was determined to calculate the amount of conjugated peptide produced. Levels of serine, threonine, tryptophan.
methionine.
* SEP-PAKs - trade-mark * Sephadex - trade-mark 'O 94/26306 ~ PCT/US94104853 tyrosine and cysteine were :not determined as these amino acids are modified under the standard hydrolysis conditions.
Example 3 5 Binding of Anti-thyroxin Monoclonal Antibodies to HCV E2HV Domain Peptides Monoclonal antibodiies that bind to thyroxine (T4) were prepared by Dr.
Mario Geysen, Chiron Mimitopes Ltd, Australia. The binding of these antibodies to overlapping peptides that: span the F,2HV region was assessed. Peptides on pins 10 were prepared essentially a;s described above in Example 2, except that the HCV
E2HV sequence spanned from amino acid 383 to 413 of HCV 1. The binding of the anti-T4 monoclonal antibodies to the HCV E2HV mimitopes was performed in duplicate. The binding results are shown in the bar graph in Fig. 6, where the solid and shaded bars represent binding of each of the duplicate samples. As seen 15 in the figures, the anti-T4 antibodies were immunologically reactive with epitope(s) encompassed within the HCV 1 sequence that spanned from aa395 to aa407.
Example 4 20 Binding of Serum Proteins to HCV
The binding of thref: serum proteins, human prealbumin, human serum albumin, and thyroid binding globulin (TBG) to overlapping peptides spanning E2HV was performed. Octamer-bearing pins were prepared as described in Example 1. The binding of the designated serum proteins to the octamers was 25 determined by an ELISA a;~say, using antibodies directed to the specific proteins.
Controls were run in the absence of the serum proteins but in the presence of the respective antibodies. The results, shown as difference plots, are shown in Fig. 7.
Based upon the results, it appears that transthyretin binds to at least one minimum epitope in the hypervariabl~~ region. Yn addition, the results are suggestive that 30 TBG binds to two minimurn epitopes, one of which encompasses the SLF--G
motif.
Claims (9)
1. An immunogenic polypeptide which is a 31 mer having the consensus sequence .T.VTGG.AARTT.G..SLF..G.SQ.IQLI derived from the E2HV hypervariable region of HCV
or a truncated version thereof which retains the SLF..G motif or variants thereof which retain the SLF..G motif and wherein at one or more amino acid residue positions the amino acid residue is substituted by another amino acid residue or by a non-naturally occurring analogue thereof, whereby immunoreactivity of said truncated versions or variants is maintained.
or a truncated version thereof which retains the SLF..G motif or variants thereof which retain the SLF..G motif and wherein at one or more amino acid residue positions the amino acid residue is substituted by another amino acid residue or by a non-naturally occurring analogue thereof, whereby immunoreactivity of said truncated versions or variants is maintained.
2. An immunogenic polypeptide as claimed in claim 1 which is a 31 mer having the sequence .T.VTGG.AARTT.G..SLF..G.SQ.IQLI
or a variant thereof wherein at one or more amino acid residue positions the amino acid residue is substituted by a non-naturally occurring analogue whereby immunoreactivity of said variant is maintained.
or a variant thereof wherein at one or more amino acid residue positions the amino acid residue is substituted by a non-naturally occurring analogue whereby immunoreactivity of said variant is maintained.
3. An immunogenic polypeptide as claimed in claim 1 which is a 31 mer having the sequence .T.VTGG.AARTT.G..SLF..G.SQ.IQLI.
4. An immunogenic polypeptide as claimed in any one of claims 1 to 3 which is coupled to a carrier protein.
5. The use of an immunogenic polypeptide as claimed in any one of claims 1 to 4 for treating an individual for hepatitis C virus (HCV) infection.
6. A composition comprising an immunogenic polypeptide as claimed in any one of claims 1 to 4 together with a pharmaceutically acceptable carrier.
7. The use of a composition as claimed in claim 6 for treating an individual for HCV infection.
8. An immunogenic polypeptide according to any one of claims 1 to 3 which is immobilized an a solid substrate.
9. An ummunoassay method far detecting anti-hepatitis C virus (HCV) antibodies in a biological sample, the method comprising the steps of:
(a) incubating an antibody - containing biological sample suspected of containing anti-HCV antibodies with an immunogenic polypeptide as claimed in any one of claims 1, 2, 3 and 8 to permit the formation of an antibody-antigen complex and (b) detecting whether any antibody-antigen complex containing said immunogenic polypeptide is formed.
(a) incubating an antibody - containing biological sample suspected of containing anti-HCV antibodies with an immunogenic polypeptide as claimed in any one of claims 1, 2, 3 and 8 to permit the formation of an antibody-antigen complex and (b) detecting whether any antibody-antigen complex containing said immunogenic polypeptide is formed.
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US6169993A | 1993-05-12 | 1993-05-12 | |
US08/061,699 | 1993-05-12 | ||
PCT/US1994/004853 WO1994026306A1 (en) | 1993-05-12 | 1994-05-03 | Conserved motif of hepatitis c virus e2/ns1 region |
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CA2162557A1 CA2162557A1 (en) | 1994-11-24 |
CA2162557C true CA2162557C (en) | 2004-09-28 |
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CA002162557A Expired - Fee Related CA2162557C (en) | 1993-05-12 | 1994-05-03 | Conserved motif of hepatitis c virus e2/ns1 region |
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EP (2) | EP0697888B1 (en) |
JP (3) | JPH08510240A (en) |
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CA (1) | CA2162557C (en) |
DE (1) | DE69433160T2 (en) |
WO (1) | WO1994026306A1 (en) |
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DE69433160T2 (en) * | 1993-05-12 | 2004-07-08 | Chiron Corp. (N.D.Ges.D. Staates Delaware), Emeryville | Preserved motif of the hepatitis C virus E2 / NS1 region |
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US6110465A (en) * | 1995-06-07 | 2000-08-29 | The United States Of America As Represented By The Department Of Health And Human Services | Nucleotide and deduced amino acid sequences of hypervariable region 1 of the envelope 2 gene of isolates of hepatitis C virus and the use of reagents derived from these hypervariable sequences in diagnostic methods and vaccines |
KR970065713A (en) * | 1996-03-19 | 1997-10-13 | 성재갑 | Secretory envelope proteins 1 and 2 of hepatitis C virus (Japanese type) |
GB9810756D0 (en) * | 1998-05-19 | 1998-07-15 | Angeletti P Ist Richerche Bio | Mimotopes of hypervariable region 1 of the e2 glycoprotein of hcv and uses thereof |
AU7602800A (en) * | 1999-09-23 | 2001-04-24 | Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services, The | Hepatitis c virus envelope two protein (e2) which lacks all or part of the hypervariable region one (hvr1), corresponding nucleic acids, chimeric viruses and uses thereof |
AU772617B2 (en) * | 1999-11-19 | 2004-05-06 | Csl Limited | Vaccine compositions |
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US20110097332A1 (en) * | 2007-08-06 | 2011-04-28 | Martina Buck | Composition and Method of Use for HCV Immunization |
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WO2010148117A1 (en) | 2009-06-17 | 2010-12-23 | Scantibodies Laboratory, Inc. | Therapeutic and diagnostic affinity purified specific polyclonal antibodies |
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GB201415714D0 (en) * | 2014-09-05 | 2014-10-22 | Medical Res Council | Antibodies and antigen binding fragments thereof |
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1994
- 1994-05-03 DE DE69433160T patent/DE69433160T2/en not_active Revoked
- 1994-05-03 WO PCT/US1994/004853 patent/WO1994026306A1/en active IP Right Grant
- 1994-05-03 CA CA002162557A patent/CA2162557C/en not_active Expired - Fee Related
- 1994-05-03 AT AT94915985T patent/ATE249838T1/en not_active IP Right Cessation
- 1994-05-03 EP EP94915985A patent/EP0697888B1/en not_active Revoked
- 1994-05-03 EP EP03077917A patent/EP1421951A3/en not_active Withdrawn
- 1994-05-03 JP JP6525508A patent/JPH08510240A/en active Pending
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1995
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- 1995-05-09 US US08/438,182 patent/US20030017156A1/en not_active Abandoned
- 1995-05-09 US US08/438,183 patent/US7135185B1/en not_active Expired - Lifetime
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1997
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2004
- 2004-11-30 JP JP2004347900A patent/JP2005097319A/en not_active Withdrawn
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2005
- 2005-05-25 JP JP2005152406A patent/JP2005325126A/en not_active Withdrawn
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- 2006-09-18 US US11/522,614 patent/US7371386B2/en not_active Expired - Fee Related
Also Published As
Publication number | Publication date |
---|---|
EP0697888A1 (en) | 1996-02-28 |
DE69433160T2 (en) | 2004-07-08 |
JPH08510240A (en) | 1996-10-29 |
US7098303B1 (en) | 2006-08-29 |
CA2162557A1 (en) | 1994-11-24 |
ATE249838T1 (en) | 2003-10-15 |
US20030017156A1 (en) | 2003-01-23 |
EP1421951A2 (en) | 2004-05-26 |
US6692907B1 (en) | 2004-02-17 |
EP1421951A3 (en) | 2005-10-05 |
US7371386B2 (en) | 2008-05-13 |
JP2005325126A (en) | 2005-11-24 |
JP2005097319A (en) | 2005-04-14 |
US7135185B1 (en) | 2006-11-14 |
EP0697888B1 (en) | 2003-09-17 |
WO1994026306A1 (en) | 1994-11-24 |
US7252827B1 (en) | 2007-08-07 |
DE69433160D1 (en) | 2003-10-30 |
US20070014813A1 (en) | 2007-01-18 |
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